key: cord-318239-2sraqm6e authors: Phan, Lan T.; Nguyen, Thuong V.; Huynh, Loan K.T.; Dao, Manh H.; Vo, Tho A.N.; Vu, Nhung H.P.; Pham, Hang T.T.; Nguyen, Hieu T.; Nguyen, Thuc T.; Le, Hung Q.; Nguyen, Thinh V.; Nguyen, Quan H.; Huynh, Thao P.; Nguyen, Sang N.; Nguyen, Anh H.; Nguyen, Ngoc T.; Nguyen, Thao N.T.; Nguyen, Long T.; Luong, Quang C.; Cao, Thang M.; Pham, Quang D. title: Clinical features, isolation, and complete genome sequence of severe acute respiratory syndrome coronavirus 2 from the first two patients in Vietnam date: 2020-05-28 journal: J Med Virol DOI: 10.1002/jmv.26075 sha: doc_id: 318239 cord_uid: 2sraqm6e In January 2020, we identified two SARS‐CoV‐2 infections in a familial cluster with one person coming from Wuhan, China. The complete genome sequences of two SARS‐CoV‐2 strains isolated from these patients were identical and 99.98% similar to strains isolated in Wuhan. This is genetically suggestive of human‐to‐human transmission of SARS‐CoV‐2 and indicates Wuhan as the most plausible origin of the early outbreak in Vietnam. The younger patient with a mild upper respiratory illness and a brief viral shedding, while the elderly with multi‐morbidity had pneumonia, prolonged viral shedding, and residual lung damage. Evidence of nonsynonymous substitutions in the ORF1ab region of the viral sequence warrants further studies. This article is protected by copyright. All rights reserved. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent In this report, we describe clinical features, virus isolation, and complete genome sequences from the first two SARS-CoV-2 infections in Vietnam. In response to the outbreak of a novel coronavirus first reported in December 2019, on 21 January 2020, Vietnam's Ministry of Health (MoH) published its interim guidelines on the surveillance, prevention, and control of Covid-19, which mandates all health facilities to report any suspected and confirmed Covid-19 cases to the MoH within 24 hours. A suspected case was defined as a person with an acute respiratory tract illness, who reported that he/she had fever and cough (with and without difficulty in breathing) and that within 14 days before the onset of disease, he/she had returned or came from areas where SARS-CoV-2 was spreading or had exposed to a laboratory-confirmed case of Covid-19. Laboratory confirmation required detection of SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swab specimens by real-time reverse-transcription-polymerase-chain-reaction (RT-PCR) assays. Apart from the respiratory specimens, blood specimens also were collected from suspected cases during acute and recovery phases of illness for serological assessment of SARS-CoV-2 antibodies. We identified a familial cluster of two patients with SARS-CoV-2 infection at a referral hospital in Ho Chi Minh City in southern Vietnam through the notifiable communicable disease surveillance system. Demographic, epidemiological, and clinical data were collected through interviews with the patients and their relatives, and a review of medical records. NP and OP swab specimens of the patients were collected in a single tube containing 3mL virus transport media and tested for SARS-CoV-2 by real-time RT-PCR assays described previously. 5 The limits of detection of the assays for the E and RdRp genes were reported as low as 3.9 copies and 3.6 copies This article is protected by copyright. All rights reserved. per reaction, respectively. We performed both the E and RdRp gene assays for confirmation of the SARS-CoV-2 infection and the RdRp gene assay for treatment follow-up. The E or RdRp gene assay was considered positive if the cycle threshold (Ct) value was less than 40. Written informed consent was obtained from the two patients. After being confirmed positive for SARS-CoV-2, NP and OP swab specimens, which were taken from the two patients on admission and during the treatment follow-up period, were stored at -70°C at the Pasteur Institute of Ho Chi Minh City until used. After thawing the specimens, virus isolation was performed using the Vero E6 cell line. In brief, Vero cells were inoculated with 400 µL of filtered specimens in 25-cm 2 flasks, containing each 5 mL of maintenance media. To increase the chance of virus isolation, we used two maintenance media: Dulbecco's modified Eagle's media (DMEM) supplemented with 2% fetal bovine serum (FBS) and DMEM supplemented with 0.2% bovine serum albumin (BSA) and 16 mg/mL N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin. 6 We monitored the flasks for the formulation of cytopathic effect (CPE) at 48, 60, and 72 hours post-inoculation. Once the CPE was observed under the microscope, cell culture supernatants were harvested, divided into aliquots, tested for the presence of SARS-CoV-2 by real-time RT-PCR assays, and stored at -70°C until virus titration and sequencing were performed. We performed virus titration on Vero E6 cells by using the standard plaque assay. 7 The virus titer was summarized as plaque-forming units (PFU) per milliliter. This article is protected by copyright. All rights reserved. We synthesized complementary DNA and used random primers for amplification. We then performed next-generation sequencing analysis with complete genome sequences of isolates generated by the MiSeq platform (Illumina, San Diego, CA). 8, 9 The consensus genome sequences were generated and mapped to the reference genome of SARS-CoV-2 (MN975262.1) using CLC Genomics Workbench version 10.1.1. We then aligned our complete genome sequences with other related coronavirus sequences archived from GenBank/GISAID using Mafft software version 7.452. Estimation of the best fitting substitution model (GTR+G+I) and inference of the phylogenetic tree were conducted by a maximum likelihood approach using MEGA-X software version 10.0.5 with 1000 bootstrap replicates. Viral sequences were classified as the method of Forster and his colleagues. 10 Furthermore, the sequences were also analyzed to identify nucleotide variants, which were then translated into amino acid variants, and compared with the Wuhan strain (MT019529). These variations were characterized as synonymous and nonsynonymous amino acid substitutions. The complete genome sequences were deposited to GenBank under accession numbers MT192772 and MT192773. Table 1 ). By the first week of hospitalization, the thrombocytopenia resolved, while the patient continued to present with lymphopenia. There were signs of disease progression, including cough with sputum, crackles over the left lung, hypoxemia since day three of hospitalization, and several pulmonary infiltrates on chest radiographs. The patient was given oseltamivir, several broad-spectrum antibacterials (including ceftriaxone, cefoperazone-sulbactam, levofloxacin, linezolid, and imipenem-cilastatin), drugs for hypertension and diabetes, and supplementary therapies. During treatment, he was also requested to use chlorhexidine mouthwashes two to six times a day; however, the compliance was poor due to dyspnea. Oxygen supplementation was begun since day four because of an occurrence of respiratory failure. Fever resolved on day four and clinical improvement was observed from day five onwards. Viral RNA was detected in respiratory specimens for up to 18 days post-admission, with a Ct range of 26.89-This article is protected by copyright. All rights reserved. 32.87 (Fig. 1A) . At the time of hospital discharge on February 12 (day 22), residual lung abnormalities remained to observe on chest x-rays. On contrary, his wife, who accompanied him on the trip to Vietnam, had no signs and symptoms of Covid-19 during the 2-week quarantine period and her NP and OP swab specimens collected on The lowest virus dilution for a visible plaque in a single field of view under the microscope was 10 -5 dilution of the stock virus from Patient 2 (Fig. 1D) , which corresponds to the viral titer of 9.5×10 6 PFU/mL. The viral titer of the stock virus from Patient 1 was 3.3×10 2 PFU/mL The length of genome sequences of viral strains isolated from the two patients were 29,891 bp and 29,890 bp with no gaps and high coverage (664× and 1,897×, respectively). Phylogenetic analyses revealed that the strains isolated in Vietnam belonged to Betacoronavirus B type and shared 99.98% sequence similarity at the nucleotide level with the strain isolated from patients in Wuhan (MT019529). It has >96.11% and 90.56% similarity with SARS-CoV isolated from bat (MN996532) and pangolin (EPIISL410721) (Fig. 2) , respectively. The full genome sequence from Patient 2 was identical to that of Patient 1, genetically indicative of human-to-human transmission. Vietnam. We thank Dr Tu Ngoc Le for medical record reviews. We acknowledge the authors, the originating and submitting laboratories of the GISAID sequences. We This article is protected by copyright. All rights reserved. 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