Eleven COVID-19 inpatients in Meizhou People's Hospital and confirmed to be infected with SARS-CoV-2 by Meizhou Center for Disease Control and Prevention (CDC) from January to May 2020. Of the 11 patients, six cases developed fever, ten cases developed cough, and two cases developed headache and chills. There were five patients with fever and cough at the same time, five patients with cough only, and one patient with fever only. Four patients (36.4%) had underlying diseases, including hypertension, diabetes, coronary heart disease and fatty liver. Pneumonia was the most common complication. In addition, one case (Case 4) developed acute respiratory distress (ARDS) and respiratory failure. All patients were treated with anti-viral therapy (Arbidol, Lopnave/litonwe, Prezista, Ribavirin and Interferon), and 1 patient was treated with non-invasive mechanical ventilation. Cases 4 and 6 were treated with corticosteroids. Cases 1, 2, 3, 4, 5, 6, 7, 8 and 9 were treated with immunoglobulin (Table 1).
Complete blood count and blood biochemistry were performed at the earliest time of admission for each patient. The laboratory test results showed that lymphocytopenia (LYM) and lymphocytic percentage (LYM%) were decreased or normal among adults, there was no adult patients with increased LYM/LYM%. Most patients had normal total protein (TP) and albumin (ALB), but only two patients had decreased. Most patients had increased or normal levels of erythrocyte sedimentation rate (ESR), C reactive protein (CRP), activated partial thromboplastin time (APTT), fibrinogen (FIB), creatine kinase isoenzymes (CK-MB), and lactate dehydrogenase (LDH), there was no patients with decreased ESR, CRP, APTT, FIB, CK-MB, LDH level(Table 2).
SARS-CoV-2 virus real-time PCR cycle threshold (Ct) value is reciprocal to virus load. It can indirectly reflect the severity of the infection. Spearman method was used to further analyze the correlation between Ct value (viral load) of SARS-CoV-2 virus and biochemical and clinical indicators. It was found that NEU (r=0.664, P=0.026), CK-MB (r=0.655, P=0.029), BUN (r=0.682, P=0.021) and SARS-CoV-2 viral load were significantly correlated (Figure 1).
According to previous research, the SARS-CoV-2 viral genome sequence was 29.9 kb long, including ten ORFs. The first ORF (ORF1ab) accounts for about 71% of the entire genome, while the remaining ORF codes for helper proteins and structural proteins. The four major structural proteins are spike glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N)[17] (Figure 2A). We identified two different SNPs at positions 8781 and 28144 (based on the reference genome NC_045512.2, SARS-CoV-2 isolate Wuhan-Hu-1) according to multiple sequence alignment (MSA). The two most variable located in the ORF1ab and in ORF8. Position 8781 located in ORF1ab gene, appears either T (U) or C variation. It causes a synonymous mutation of amino acids at position 2839 (p.Ser2839Ser). It is likely not to introduce phenotypical differences between the different strains. In addition, position 28144 located in ORF8 gene and appears either T (U) or C variation. It causes a Ser/Leu change in amino acid 9214 (p.Ser9214Leu), which Serine is a polar amino acid and Leucine is nonpolar. This may cause conformational changes in amino acid peptide chains, given that Serine is a polar amino acid, and Leucine is nonpolar. According to the sequences of the 34 viruses we analyzed, if it was a T base at position 8781, it must be a C base at position 28144. In contrast, if there is a C base at position 8781, there must be a T base at position 28144 (Figure 2B). The two SNPs were found to maybe have a completely linked genetic form of 8781C-28144T and 8781T-28144C.
We sequenced the virus of the first patient (case 1) admitted to our hospital using Sanger sequencing and the sequencing result was compared with the sequences from GenBank. The results suggested that case 1 also had variation at positions 8781 and 28144. We sequenced virus samples from other ten COVID-19 patients admitted to our hospital for two different SNPs. The results showed that case 1, 2, 6, 7, 8 and 9 was T base at position 8781 and C base at position 28144, and the C base at position 8781 and the T base at position 28144 for cases 3, 4, 5, 10 and 11 (Figure 3A). We also attached the Sanger sequencing results (Figure 3B). These sequences have been deposited to the GenBank database. And GenBank accession numbers are MT856370, MT856692, MT872190, MT872188, MT872187, MT872189, MT510727, MT860726, MT510728, MT872199, and MT872198 for position 8781. GenBank accession numbers are MT856370, MT856443, MT860461, MT860462, MT860463, MT860464, MT510727, MT856477, MT510728, MT860465, and MT860469 for position 28144.