key: cord-1052501-a5ypvv4f authors: Ichikawa, Takaya; Torii, Shiho; Suzuki, Hikoyu; Takada, Akio; Suzuki, Satoshi; Nakajima, Masahide; Tampo, Akihito; Kakinoki, Yasutaka title: Mutations in the non-structural proteins of SARS-CoV-2 may contribute to adverse clinical outcome in COVID-19 patients date: 2022-05-10 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.05.010 sha: 8200a280f18acd94d521041788d9745ecb182797 doc_id: 1052501 cord_uid: a5ypvv4f Background . From late March through April 2021, we experienced a cluster of COVID-19 patients, named “Cluster K”, with rapid severe illness compared to those who were infected before. Method . COVID-19 patients enrolled in this study were divided into two groups: 66 patients from November 2020 to March 2021(group A), 37 patients whose infection links were traced from Cluster K (group B). The primary outcome was mortality rate, and the secondary outcome was maximal oxygen flow rate as severity of the disease. Viral genome sequences were compared between two groups. Results . Mortality rates were 6.1% in group A and 16.2% in group B (OR: 2.97, 95%CI: 0.65-15.38). The patients in group B required high oxygen flow rate (O2 ≥10 L/min) in earlier clinical course (p=0.029). Viral genome sequences revealed five amino acid mutations. Of these, four were found on three non-structural proteins (NSPs): one in nsp3 and 15, two in nsp6 (one of them is near the potential sites under positive selective pressure). Other one was on S protein. Conclusion . This study suggests that mutations in NSPs, especially nsp6, are associated with adverse clinical outcome in COVID-19 patients. The Coronavirus disease 2019 , which is caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initially broke out in Wuhan, China, on December 2019 and has subsequently spread around the world (Gorbalenya et al., 2020) . SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA genome of approximately 30 kb. Two-thirds of the viral genome at the 5" terminus contains ORF1a and ORF1b, which encodes 16 non-structural proteins (NSPs). These NSPs play crucial roles in viral replication and evasion of host immune systems (Maier et al., 2015) . The other one-third of the 3" end contains structural genes such as spike glycoprotein (S protein), envelope protein, membrane protein, nucleocapsid protein, and several accessory proteins (Hagemeijer et al., 2010) . Because replication of RNA viruses typically has a low fidelity, the viral genome of SARS-CoV-2 has accumulated mutations at an average of two nucleotides per month (Duchene et al., 2020) . On 25th February 2021, World Health Organization (WHO) defined a variant of interest (VOI) as an isolate detected in several countries that changes phenotype under certain conditions, and a variant of concern (VOC) as an isolate in VOIs if it has been demonstrated to be associated with increase in transmissibility or virulence (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). For example, VOC-202012/01 (Pango lineage B.1.1.7), as it is called "alpha variant", has higher transmissibility and mortality compared to a reference isolate (Challen et al., 2021; Davies et al., 2021) . These variants, however, were not focused on mutations in NSPs, but in structural proteins, especially S protein. It remains unclear whether mutations in NSPs are responsible for the virulence of COVID-19. As of April 2022, COVID-19 is responsible for 7.1 million cases and 29 thousand deaths in Japan (https://www.mhlw.go.jp/stf/covid-19/kokunainohasseijoukyou.html). The most remarkable clinical feature of this disease is its heterogeneity in clinical manifestations, ranging from no symptoms to critical illness (Grasselli et al., 2020) . Several risk factors for symptom severity have been known, such as age (over 65 years old), male sex, obesity, history of smoking, and comorbidities (including hypertension, diabetes mellitus, respiratory disease, cardiovascular disease, chronic kidney disease and cancer) (Gansevoort and Hilbrands, 2020; Liang et al., 2020; Popkin et al., 2020; Zheng et al., 2020) . In Japan, the several treatment options are currently recommended based on the We found that the progression of disease among the patients in this cluster were faster compared to patients who were infected before that. Therefore, in order to identify factors associated with the progression of disease, we examined the viral genome sequences along with the clinical characteristics and treatment strategies, and then compared those with other cases. All adult patients with COVID-19 who were admitted to Asahikawa City Hospital from November 2020 to April 2021 were enrolled in this retrospective study. All positive results were confirmed by real-time RT-PCR for the presence of SARS-CoV-2 in saliva or nasopharyngeal swab samples. The recovered patients were discharged from the hospital following the discharge criteria provided by Japanese Ministry of Health, Labour and Welfare when two criteria have met:10 days passed from the symptom onset and 72 hours passed from the symptom resolution, or when two consecutive negative PCR results are confirmed. This study was approved by the ethics committee of Asahikawa City Hospital (approval number 5, 2021). The opt-out method was used to obtain patient consent in this study. We provided the patients with information explaining the proposed research plan via the website of Asahikawa City Hospital. The epidemiological data, medical history, underlying comorbidities, symptoms and signs at admission, oxygen flow rate, treatment and clinical outcomes were obtained from electronic medical records. Because the date of onset was unclear for some patients who were asymptomatic, we considered the date on which positive PCR result was obtained as day 0. Clinical outcomes were followed up until discharge or death. The primary outcome was mortality rate, and the secondary outcome was severity of the disease stratified by maximal oxygen flow rate into O2 ≥1 L/min, ≥5 L/min and ≥10 L/min. The days to reach its oxygen dose was calculated as occurrence of an event, and cumulative probability was plotted using Kaplan-Meier method. The stored RNA extracts were used as clinical samples. Fist-strand cDNA was synthesized by using a PrimeScript IV 1 st strand cDNA Synthesis Mix ( TaKaRa Bio) with random hexamer primers and extracted RNA, according to the manufacture"s protocols. The whole-genome sequencing of SARS-CoV-2 was carried out as reported previously (Torii et al., 2021) . Briefly, a total of nine gene fragments shown in Figure S1 were amplified with synthesized cDNA, specific primer sets for SARS-CoV-2 and PrimeSTAR GXL DNA polymerase (TaKaRa Bio). Then, the amplified products were directly sequenced in both directions by using the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) with specific primers. The primer set using in this study are listed in Table S1 . Our raw sequence data were converted to FASTQ format by in-house Python script with BioPython module (Cock et al., 2009) More than one million SARS-CoV-2 published genome sequences whose length were between 29,000 -30,000 bps were downloaded from National Center for Biotechnology ORFs were less than 100, all non-synonymous mutations which characterize each variant were included, and all other substitutes observed in more than 95% of the samples that satisfy the above conditions were also included. The non-synonymous mutations which characterize each variant are shown in Table S2 , created based on the mutation prevalence information from outbreak.info (https://outbreak.info/). ORF10 was excluded from the analyses because one of our assemblies did not completely cover that region. Based on the alignment, suitable nucleotide substitution models for each partition were selected by ModelTest-NG (Darriba et al., 2020), followed by maximum likelihood phylogenetic analyses using the selected substitution models by RAxML-NG (Kozlov et al., 2019) . The constructed phylogenetic tree was drawn and edited by MEGA X (Kumar et al., 2018) . We performed group comparisons using Fisher"s exact test for categorical variables, and independent group t-test for continuous variables. The categorical variables were expressed as frequencies (percentage), and the continuous variables as the median with interquartile range. Odds ratios (OR) with 95% confidence interval were calculated for the rates of mortality and the required oxygen flow rate. Cumulative probabilities of oxygen therapy were described with using the Kaplan-Meier analysis and compared using log-rank test. P-values of 0.05 or less were considered statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan, version 1.54) (Kanda, 2013) , which is a graphical user interface for R software (The R Foundation for Statistical Computing, Vienna, Austria). A total of 191 patients diagnosed with COVID-19 were admitted to our hospital from November 2020 to April 2021. The patients were divided into two groups, group A: the patients infected from November 2020 to March 2021, and group B: the patients whose infection links were traced from Cluster K. We included the patients whose ages range between 65 and 89 years old for the following reasons; 1) patients who are younger than 65 years old are not necessarily requested for hospitalization in our city"s health care system, 2) in group A, many patients over 90 years old were transferred from a convalescent hospital where COVID-19 outbreak had occurred, thus the age profiles would be much different from group B if the patients aged over 90 years old were included in this group. Finally, 66 patients were assigned to group A and 37 patients to group B (Figure 1 ). Table S3 . Because no significant difference was found in mortality rate, severity of the disease stratified by maximal oxygen flow rate was evaluated as a secondary outcome. Cumulative probability of the patients who required low dose oxygen therapy (O2 ≥1 L/min) were similar in both groups (Table 3, Figure 2A ). However, the patients in group B were likely to require more oxygen therapy in earlier course of hospitalization ( Figure 2B, 2C) . For high dose oxygen therapy (O2 ≥10 L/min), the significant difference was observed (p=0.029). As with mortality, males in group B showed worse outcomes in the severity of the disease (Table 3 ). Assuming that these differences in clinical outcome would be due to viral factors, we examined the whole viral genome sequences representing two samples in each group, named Asahikawa_0108 and Asahikawa_0122 isolated in January 2021, Asahikawa_0404 and Asahikawa_0417 isolated in April 2021. The mutation sites which differed in these four samples are summarized in Table 4 . Focusing on the non-synonymous mutations, five amino acid changes were identified (represented in bold). Of these, four amino acid changes were found on NSPs and one was on S protein, as illustrated in the viral genome scheme of SARS-CoV-2 ( Figure 3 ). In order to assess strain relatedness, the phylogenetic analysis was carried out. The isolates in this study were closely related to the strain previously isolated in Japan (accession number BS000756.1; Pango lineage B.1.1.214), but not to any of the variants labeled by WHO ( Figure 4 ). We here experienced the cases of a single COVID-19 cluster in which the conventional treatment was unsuccessful and the hospitalized patients became more severely ill, thus performed the retrospective analysis to find out the causative factors. The patients in group B had a higher mortality rate compared to group A, but no significant difference was indicated. For the severity of disease, the patients in group B required higher oxygen flow rate in early course of admission. Furthermore, the differences became larger when limited to males, with 41.2% of male severe patients in group B against 6.3% in group A. In terms of treatment strategies, we were likely to use methylprednisolone monotherapy for severe cases in group B. This was because we had experienced that, in the winter of 2020, patients who received methylprednisolone therapy appeared to have a better response than those who were treated with remdesivir after poor response for dexamethasone, although the treatment guideline for COVID-19 in Japan recommends the use of remdesivir for patients with pneumonia whose oxygen saturation falls below 96% (https://www.mhlw.go.jp/content/000815065.pdf). In this study, similar results were obtained when the secondary outcome was compared only for patients without remdesivir (data not shown). Therefore, the viral factors rather than the difference of the treatments could have an impact on the clinical outcome. Whole viral genome sequences revealed five non-synonymous mutations by comparison of the isolates with each group. Interestingly, four of them were found on NSPs. The sole structural mutation, D1153G on S protein, was located near the C-terminus and outside the range of three-dimensional structures obtained by crystallography, suggesting neither the head nor stalk of S protein. Therefore, D1153G is not expected to involve in the virulence of the disease. Of the four mutation sites in NSPs, two were found on nsp6 (T3598I and Y3722C), and the rest of them were on nsp3 (E2089D) and nsp15 (A2143S). To our knowledge, none of these mutations are known to be the factors that determine severity of the disease in clinical practice, and it is unclear whether the mutations affect the functions of these NSPs. However, all of them have been reported to play roles in suppression of type 1 interferon in host cells (Shemesh et al., 2021; Shin et al., 2020; Xia et al., 2020; Yuen et al., 2020) , and also nps6 involves in the formation of autophagosome in host cells. The potential sites under positive selective pressure have been found on nsp6 near T3598I according to an evolutionary analysis on SARS-CoV-2 genome sequences of 351 clinical samples (Benvenuto et al., 2020) . Therefore, the mutations in nsp3, 6 and 15 may be responsible for its function to interact with host immunity and autophagy. In particular, nsp6 is known to interact with Sigma-1 receptor, which is a transmembrane endoplasmic reticulum protein that modulates activity of multiple effector proteins, and is a potential target for antiviral drugs (Gordon et al., 2020a) . Sigma-1 receptor (Gordon et al., 2020b) , suggesting that nsp6 could play an important role clinically. Further investigations are required to reveal their functions and involvement in the virulence. The phylogenetic analysis revealed that the isolates in this study are derived from Pango lineage B.1.1.214 which was the dominant strain in so-called "the third wave", the epidemic period from October to December 2020 in Japan (National Institute of Infectious Diseases, 2021). Considering the fact that any strains identical to the viral sequence of group B cannot be found in more than 1,000,000 registered sequences in NCBI, it is likely that the virus evolved locally from January to April 2021 in Hokkaido. Authors have no conflicts of interests. The use of patient"s clinical information was approved by the Research Ethics Committee of Asahikawa City Hospital which oversaw the study conduct and documentation. This study was conducted in accordance with the principles of the Declaration of Helsinki. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors included in my manuscript declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Position number based on alignment to NCBI reference sequence: NC_045512 (Wuhan-Hu-1). The different parts between the two samples in group A are shown in a gray area. Amino acid changes identified between two groups were represented in bold. 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