Comparison of the critical virulence-associated residues and geophylogenetic associations
Nonstructural protein 1(nsp1) in betacoronaviruses is a major virulence factor along with nsp3, nsp7, nsp17, ORF3a, E protein, M protein, and the nucleocapsid protein. Nsp1 virulence is expressed as an IFN antagonist, inhibitor of cell cycling and apoptosis, and an inhibitor of both virus- and IFN-dependent signaling.
Residues that were found to inhibit both antiviral signaling and gene expression in SARS are marked with red box above in the multiple sequence MUSCLE analysis (Jauregui et al 2013; Wathelet et al 2007). Residues 1 to 12 and 129 to 179 are flexibly disordered. These virulence-associated residues (E41, R43, Q44, K47, H81, H83, E91, E93) were poorly conserved in MERS (0% in critical virulence residues) except for electrostatic covariance in Arg43->K43 and K46-R46 (overall %ID 21.7% for residues 13-127).
The phylogenetic tree of 235 nsp1 peptide sequences (residues 13-127) was generated with MEGA7 using Maximum Likelihood and visualized in Jalview. The sequences query was the full length COIVID-19 nsp1 sequence (position 1-180). MERS nsp1 sequence was obtained separately from the initial query as there were no similarity matches due to the low overall sequence similarity with COVID-19 using BLASTp and DELTA-BLAST. The sequences were trimmed to span position 13-127 of nsp1. The evolutionary relationship with the various Bat-SARS coronavirus are easily identified. The MERS branch is interesting. For example, the evolutionary jump from SARS -> MERS may have been from Hipposideros commersoni (Nigeria|Zaria bat|ADY692.1|2008) or Hipposideros pratti|Bat-Hp-betacoronvirus|Zhejiang2013). The geographic range for Hipposideros commersoni is designated as Madagascar but the sequence is from Nigeria suggesting a novel migration pattern since the distance is 3,233 miles (near threatened A2d IUCN). The Hipposideros pratti geographic range is limited to China and Vietnam (IUCN). Memish et al in 2013 looked at 96 bats in 7 species (Rhinopoma hardwickii, Rhinopoma microphyllum, Taphozous perforatus, Pipistrellus kuhlii, Eptesicus bottae, Eidolon helvum, and Rousettus aegyptiacus) in Saudi Arabia. Only one bat, the Egyptian Tomb Bat Taphozous perforatus, had 100% nucleotide identity to the virus isolated from the human index case of MERS in 2012. The geographic range of Taphozous perforatus is widespread including Madagascar, China, and the Middle East (iNaturalist/hipposiderospratti). Using geolocation phylogeny, we might consider the geographic origin of MERS was actually in Southeast Asia. Taphozous bats are known to co-habit roosts with Hipposideros (Pavey et al 1999). Further suggesting a further need to look at cohabitation roosts as a source of interspecies transmission.
This will be updated as more information is obtained. Follow this post for further information weekly.
VIRULENCE FACTORS IN BETACORONAVIRUS
Comparative Analysis of the viral nsp1 protein
As of February 17, 2020, there have been reported 70964 cases with 1773 deaths worldwide for a case-fatality rate of 2.5%. >90% of the cases and reported deaths are from mainland China. In the USA, 34 cases have now been confirmed.
Despite the concern over the R0 of 3-5.5, the virulence of the COVID-19 is much lower than SARS and MERS. We are lacking in the comparison between these viral species. Thus is the question we are asking-What is the difference in the virulence-associated residues that might account for the observed differential virulence?
A review of the literature identified 7 major virulence associated viral peptide: the nonstructural proteins nsp1, nsp3, nsp7, ORF3a, and the E,M, and N structural proteins.
MERS is distant phylogenetically from COVID-19, SARS-CoV, and all but 2 Bat-related SARS sequences
The MERS outgroup is distinctly distant from COVID-19, SARS, and except for Zaria BatCoV|ADY69162.1 and the Bat CoV Zhejiang
The nsp1 protein actions include IFN antagonism; inhibition of cell cycling and apoptosis; inhibition of gene expression and inhibits both virus- and IFN-dependent signaling. The critical residues in nsp1 that are involved in virulence are E41, R43, Q44, K47, H81, H83, E91, E93, in three distinct domains. E-41->K47 located in a distinct alpha-helix, represents the major interacting domain. The only difference is the substitution of the charged Glu44 in SARS CoV to the uncharged Gln44 residue. That is observed in the electrostatic surface model of COVID-19 with SARS (2GDT). The HGH motif is conserved structurally between SARS and COVID-19. The E91L92D93 motif in SARS is not different in sequence but is uniquely different in the COVID-19 model. Whether that contribute to the observed differential virulence remains to be studied in the lab.
Modelling using 2GDT|Chain A as the template in UCSF Chimera.
The phylogenetic evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model . The tree with the highest log likelihood (-1366.0540) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.8704). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 270 amino acid sequences (. All positions containing gaps and missing data were eliminated. There were a total of 112 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. The individual sequence data is shown below:
Minimum Sequence Length: 115
Maximum Sequence Length: 378
Average Length: 172
Trimmed nsp1 (Position 13-127)
Minimum Sequence Length: 114
Maximum Sequence Length: 124
Average Length: 116
E coli 2
The final reconstruction demonstrated the closest relatives to COVID 19 were the Bat-related SARS AVP78030.1|Rhinolophus sinicus|Zhoushan City|China|2017, AVP78041.1|Rhinolophus sinicus|Zhoushan City|China|2015 and a second branch containing BatCoV YP_003858583.1|Rhinolophus species|Kenya|2007 and the Bat-related SARS APO40578.1|Rhinolophus lasii|Bulgaria|2008. This would suggest that COVID-19 evolved from a bat coronavirus and in circulation since 2015.
MERS is distantly related to both COVID-16, SARS, and Bat-related SARS nsp1 sequences with the notable exception of 2 Bat-SARS sequences below from Nigeria and China:
>BatCo|ADY69162.1:1-171 putative 1a polyprotein, partial [Zaria bat coronavirus]
>BatCoVZhejiand2013|YP_009072438.1:8-174 non-structural polyprotein [Bat Hp-betacoronavirus/Zhejiang2013]
It is possible these last two sequences may provide a link to a previously unreported MERS spillover from MERS-related batCoV viruses.
The MERS outgroup also contained all ErinaceousCoV sequences. 3 E coli RDRNAP sequences were excluded from the analysis as they were deemed to be fecal contaminants in the source data.
Summary: It appears the COVID-19 coronavirus has been in circulation since at least 2015 in the Rhinolophus sinicus bat population in China. The differential virulence in COVID-19 nsp1 nonstructural protein may be due to the charge difference in the virulence motif 1 and possibly motif 3. These will need to be studied further in the lab.
Next Week: The nsp3 macrodomain and its antagonization of IFN
OKLAHOMA ONE HEALTH INITIATIVE: SARS COV-2 IN OKLAHOMA: THE FIRST 30 DAYS
Coronavirus Outbreak in Oklahoma: The First 30 Days
Hale O’mana’o Research
We understand death for the first time when he puts his hand upon one whom we love.
~Madame de Stael
We don’t know who patient zero was, and it is very unlikely that we will ever know.
Towards the end of December of 2019, in Wuhan, China, a city of nearly 19 million people, 44 cases of pneumonia of unknown etiology were reported to the World Health Organization. Eleven became severely ill. On January 7, 2020, the National Health Commission China isolated a new coronavirus. Although the Chinese authorities initially reported no known human to human transmission, by the end of January the new coronavirus had spread to Japan, the Republic of Korea, and Thailand. On January 21, 2020, an individual who had traveled to Wuhan and returned to Washington on January 15 became ill. A clinical specimen was analyzed by the CDC and the diagnosis of a travel-related coronavirus infection was confirmed. The first human to human transmission in the US occurred on January 30, 2020, in Illinois.
On January 30, 2020, the WHO declared the coronavirus outbreak to be a Public Health Emergency of International Concern (PHEIC). In the US, CDC Director Robert Redfield stated, “Our assessment remains that the immediate risk to the American public is low.” At that time, the US had only 7 reported cases.
By February 1, 2020, this novel coronavirus had spread to 23 countries outside of China, for a total of 11,953 cases worldwide. By February 15, 218 cases outside of China were identified. That would change rapidly. By March 1, 2020, there were reported 7,169 cases and 98 deaths. 9 days later, Italy case count rose to 7,375 cases with 366 deaths. The severity of this pandemic and its toll of the human population would soon be made clear. My colleague from Italy explained their situation,
“Here the problem is mainly the lack of beds in ICUs, only 5200 scattered in all the national territory. The CFR for COVID-19 has confirmed to be not high: in Italy, we have an extremely old population (+82-year-old) and this explains the percentage points variation in deaths compared to China. Other factors for the severity of the epidemic here, more than in China, are the high number of health workers infected (here in my province, Sardinia, the 50% of infected are doctors and nurses) and some irresponsible behaviors by someone that led to super-spread events.
The lessons we've learnt so far are:
1) that the containment should be aggressive and prompt, without "steps" and hesitations, an extraordinary challenge always need extraordinary measures, the quarantine should be respected from the first till the last day;
2) the safety of the health workers is an absolute priority, this means to give the right information on all the personal protection equipment’s and especially ffp3 masks to avoid the shortage of them as it has happened here in Italy;
3) it's important to grant a bed with artificial respiration to everyone who needs it, eventually creating new ones.
4) it's not a problem of China nor of Italy, of USA, or Iran or someone else, it's not a virus of whites, yellows, blacks or greys, it's a problem of the world and only if we'll be united against it we'll win this fight.
We should fight together as humanity, not alone, or even worst, against other humans.
Please, be careful, that's not the final plague, but it kills.”
By March 7, there were 213 cases in the US and 11 deaths. One of those cases was in Tulsa County, Oklahoma, a man in his 50s who returned from Italy on Feb. 23 and began showing symptoms on Feb. 29. Three days later, another travel-related case from Italy was reported, again in Tulsa County.
On March 11, 2020, The WHO declared the coronavirus outbreak to be a pandemic. Tedros stated”: “This is not just a public health crisis; it is a crisis that will touch every sector — so every sector and every individual must be involved in the fight…we have never before seen a pandemic that can be controlled.” He was right.
When the case count for Oklahoma was 7 on March 15, Governor Stitt posted that, “the emergency declaration allows small businesses to get access to federal loans and lets state agencies hire additional staff and make purchases quicker than they would normally. The declaration also will allow Oklahoma hospitals to cut through red tape in order to treat patients faster.”
March 16. The USA morning case count was ~2000 cases and 48 deaths. By the end of day reporting, the total cases here in the US rose to a staggering 3503 and 58 deaths. We're not doing fine Oklahoma. This is a critical junction. Pay attention.
The CDC has also recommended in general that gatherings of 50 or more people over the next 8 weeks be canceled or postponed. People taking everyday steps like limiting large gatherings and frequently washing hands can substantially reduce risk around the country. Healthy individuals can also make a difference by checking in older or immunocompromised friends, family, and neighbors while maintaining physical social distancing.
On March 24, Gov. Kevin Stitt ordered that all non-essential businesses in the 19 counties that have confirmed cases to close for 21 days starting at 11:59 p.m. Wednesday. The following day on March 25, the Oklahoma State Board of Education voted Wednesday to close public schools statewide for the remainder of the school year and to turn to distance learning which up to now, was prohibited in this state. On that date, there were 165 cases and 5 deaths reported with a doubling of cases every two days.
Governor Stitt also ordered elderly and vulnerable Oklahomans to stay indoors until April 30, except for essential travel, such as to pick up groceries and prescriptions.
The next day, there were 248 cases and 7 deaths.
April 1 brought the case count in Oklahoma to 719 cases with 30 deaths. The projected “peak” calculated to be on April 22. The majority of cases occurred in Oklahoma County (Oklahoma City General area/192 cases/+37 in 24 hours), and Tulsa County (115 total cases/+32 in 24 hours). We were just entering the exponential increase with limited hospital beds and ICU beds. Our model of time-adjusted CFR remains the same at 9% in symptomatic patients, mostly male and over the age of 50. Please be compliant with the rules of quarantine. Younger age groups, as expected, are less compliant with this. We will see a greater increase in both morbidity and mortality in the next six weeks due to the limited healthcare capacity.
April 2. Today marks 3 weeks since the first case reported in Tulsa County on March 10, 2020. Since the lockdown two weeks ago, there has been some improvement in the R (0) [based on 1-week incubation period]. The R (0) 1 week ago was 6.5; it is now 3.5, within the range what we expect. We need this to drop to 1.0 (no transmission). If that remains at that rate, we may see as many as 2500 total cases at day 28.
April 3. 988 cases/38 deaths; up 109 in 24 hours 40% of which are located in Oklahoma and Tulsa counties. No real significant change in the time-adjusted CFR (9%) and R (0) (3.1). It is likely we will surpass the predicted 46 deaths in three weeks. The good news is that there are 13,000 testing kits. Most will be used for symptomatic patients. We all need to do our part with avoiding transmission to get that R (0) down to 1.
April 5. 1252 cases and 46 deaths currently. The doubling every 2 days is better so that's an improvement. Unfortunately, we hear stories of grocery stores being packed and general complacency. We really need to maintain the distance and avoid any unnecessary travel. Last week, both Oklahoma and Tulsa counties were in the top 10 nationally for people traveling outside their home. Just please limit contact with others and be mindful of physical distance. For those of us that had been exposed and tested positive, the OSMA recognizes the 3-7 back to work but they also recommended repeat swabs to be sure you are negative. The median shedding time is around 10 days. If you are still symptomatic, retest and wait 7 more days after resolution of symptoms so as to not unintentionally transmit to someone else.
April 7. Today it is reported we now have 1327 cases and 51 deaths. The hardest-hit counties are Oklahoma (279/11), Tulsa (249/8), Cleveland (180/11), Creek (45/0), Comanche (41/0), and Greer (26/3). Greer's situation is worth looking at as it's located in rural SW Oklahoma and may predict serious consequences with its 12% non-adjusted CFR. Rural Healthcare facilities are not likely capable of handling this.
Testing has also ramped up for the last two weeks. Hopefully, this will continue.
The good news is that, despite the population mobility, the R (0) is 2.8 and no doubling of cases from 4 days ago!
April 8. Our first reported case nearly one month ago was on March 7, 2020. On April 12, 2020, the OSDH has reported 1970 cases with 96 deaths (non-adjusted CFR 4.92%, time-adjusted CFR 7%.
How are we doing in this State when compared to other nations when they reported similar numbers?
Not well. As of today, 53 countries have reported ~2000 cases (plus/minus ~500) and were the basis of this report. The mean # cases were 2010.8 (standard deviation 168.7) and a mean #deaths to be 44.3 (standard deviation 42.8) resulting in an overall CFR of 2.2%. 14 countries reported a CFR of <1% (green) and 2 were >6% (red).
April 12. Day 31
How are we doing in this State when compared to other nations when they reported similar numbers?
Not well. As of the date of this analysis, 28 states have reported ~2000 cases (plus/minus ~200) and were the basis of this report. The mean # cases were 2073.1 (standard deviation 128.3) and a mean number of deaths 45.8 (standard deviation 24.3) resulting in an overall CFR of 2.2%. Only the States of Washington (5.2%) and Kentucky (5.1%) reported a higher case fatality rate at this point compared to Oklahoma at 4.9%. When we perform a time-adjusted case fatality rate (TACFR) taking into consideration when that individual death was first reported as a confirmed case, that TACFR has hovered between 6-10%. Furthermore, as of this report, there have been 457 hospitalizations and 99 deaths. Assuming all 99 deaths were also recorded as hospitalizations, that would result in a frightening 20% risk of death if the illness is severe enough to require hospitalization. We won’t have the ICU capacity to address these intensive care admissions at the current rate.
We are at a critical point in this outbreak here in Oklahoma. Do we start opening up nonessential businesses in two weeks? Personally, that might be premature since it is unlikely our peak will be reached on the projected date. Returning to work given the current CDC recommendations of 7 days after onset of symptoms and three days after resolution of symptoms may not be sufficient. Why? The median time to minimal virus titers is thought to be around 8 days. RT-PCR analysis of SARS-CoV-2 RNA and antigen is detectable up to 7 days before symptoms begin and may persist up to 28 days (37 days in another report). The duration of symptoms lasts around 14 days. The IgG antibody response is just beginning its rise for protective immunity. If we push to eliminate transmission and get the reproductive number down to R (0) =1. That would require that 21 days should pass before a confident release from quarantine since the onset of symptoms to reduce the risk of transmission to less than 90%.
We agree with the following statement from Rapid Expert Consultation on SARS-CoV-2 Viral Shedding and Antibody Response for the COVID-19 Pandemic (April 8, 2020).
Waiting for all tests to be repeatedly negative is the most conservative approach but may result in a prolonged unnecessary isolation. Assessment of humoral and cellular immune responses may also be informative.
Gaps in knowledge:
Duration of shedding of infectious virus by recovered patients and the relationship to the detection of viral RNA
Knowledge of immune mechanisms responsible for virus clearance that might predict recovery and help determine when patients are no longer infectious
Immune correlates of protection
Duration of protective immunity
We would add the following additional recommendations:
Reassess current interventions as to efficacy and incidence of adverse reactions
Promote research into effective vaccine development
Increased utilization of effective proven treatment options by clinical trial
Recognize the “second wave” of transmission (resurgence) and reassess the impact of early intervention (steroids, others) on the incidence of recurrence of COVID-19 in recovered patients
Identification of new zoonotic and ecologic niches of SARS CoV-2 to reduce risk of reemergence
Two negative repeat RT-PCR tests or 21 days following onset of symptoms or 1 week after the complete solution of symptoms before release from quarantine so that we reduce the risk of transmission to <10%.
Our physicians and all healthcare providers and staff cannot be overwhelmed at this point. We have a duty to our families, friends, and neighbors if we are to recover in a prudent fashion.
Data obtained from the following resources:
WHO SARS CoV-2 situation reports [https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports]
Johns Hopkins Coronavirus Resource Center [https://coronavirus.jhu.edu/map.html]
Coronavirus COVID-19 Data in the US repository [https://github.com/nytimes/covid-19-data
Oklahoma State Department of Health [https://coronavirus.health.ok.gov/]
Rapid Expert Consultation on SARS-CoV-2 Viral Shedding and Antibody Response for the COVID-19 Pandemic (April 8, 2020) [https://www.nap.edu/read/25774/chapter/1]
GUIDANCE ON THE ESSENTIAL CRITICAL INFRASTRUCTURE WORKFORCE [https://www.cisa.gov/publication/guidance-essential-critical-infrastructure-workforce]
IHME COVID-19 Projections [https://covid19.healthdata.org/united-states-of-america/Oklahoma]
Send comments and recommendations to the primary author @ Email: email@example.com.
The full preprint I available is now available at