Influenza-like illness caused by the 2019 novel coronavirus (2019-nCoV) via the person-to-person transmission

The ongoing pandemic of the 2019 novel coronavirus disease (COVID-19) raises a global health crisis, which has resulted in 75,778 confirmed cases with 2130 deaths in China and beyond. Atypical symptom renders it challenging to earlier recognize the 2019-nCoV carrier with the potential ability of equivalent transmission. Therefore, it is needed to gain full spectrum of COVID-19. Here we report clustered COVID-19 cases of person-to-person transmission. The symptoms of typical pneumonia are shared by the two familial members, namely son (Patient 1) and father (Patient 2). Unexpectedly, an influenza-like illness (ILI) is also caused in Patient 3 having close contact with Patient 1 at personal dinner party. Combined with clinical and epidemiological study, chest computed tomography (CT) and molecular diagnosis demonstrate that all the three cases tested positive for COVID-19 with distinct symptoms by human-to-human transmission. To the best of knowledge, it closes in part (if not all), a missing gap of clinical repertoires of COVID-19 outbreaks and underlines the possibility that neglection of cryptic/asymptomatic/mild cold-like syndromes gives biased screen in the earlier stage of COVID-19 cases.


Introduction
Like the other two outbreaks of the 2003 SARS (severe acute respiratory syndrome) in China 1, 2 and the 2012 MERS (middle eastern respiratory syndrome) in Saudi Arabia 3,4 , the 2019 novel coronavirus disease  is also a newly-emerging pandemic of global public concern 5 . The causative agent of COVID-19 (provisionally termed 2019-CoV by WHO [6][7][8] , and then renamed SARS-CoV-2 by the international Committee on Virus Taxonomy with great debate 9 ) is a new lineage within β-coronaviruses featuring with a single-stranded, positive RNA genome 6,10 . In fact, COVID-19 is initially recognized as an un-explained pneumonia in Wuhan, China in the late of December of 2019 11,12 , and then determined to be a viral respiratory illness caused by a new pathogen 2019-nCoV [6][7][8] . This agent is believed to be a zoonotic agent in that earlier cases of COVID-19 is found to display the history of close contact with "Huanan seafood wholesale wet market" in Wuhan City, China 6,8 . Extensive analysis of genomic epidemiology further suggests that all the isolates of 2019-nCoV are nearly identical, and phylogenetically-related to three bat SARS-like CoVs of bat origins 6,10,13 . Among them, bat-SL-CoVZC45 (29,802bp, Acc. no.: MG772933) and bat-SL-CoVZXC21 (29,732bp, Acc. no.: MG772934) are sampled from Rhinolophus sinicus, in Zhoushan, Zhejiang, 2017 13 , and the β-CoV/bat/Yunnan/RaTG13/2013 (Acc. no.: EPI_ISL_402131) is collected from fecal swabs of Rhinolophus affinis, in Yunnan, 2013 6 .
Worrisomely, this deadly pathogen 2019-CoV exhibits an unprecedented ability of human-to-human transmission [14][15][16] . In the past two months since its recognition of atypical pneumonia with unknown etiologic agent on Dec. 8 11,12,21 . Indeed, the confirmed 2019-CoV case differs greatly, ranging from the severe patients with severe respiratory failures 3,11 , the pneumonia of mild symptoms 14,15 , to asymptomatic carriers 20,21 . This renders the monitoring of COVID-19 clinically complicated (and/or challenging). Surprisingly, none of the accumulated 1174 cases of 2019-nCoV infections dies in Zhejiang Province, in spite that it is geographically close to Hubei Province, a centering site of this COVID-19 outbreak. It hints that local prompt administrative response plays an important role in efficient monitoring, transparent tracking, and timely supportive therapies upon the onset of suspected cases detected.
Because that current situation of COVID-19 proceeds rapidly, our understanding the dissemination and spectrum of COVID-19 remains fragmentary. Here we report sporadic cases of COVID-19 in Lishui City, Zhejiang Province, China. To the best of knowledge, we describe influenzalike illness (ILI) is caused by 2019-nCoV via the person-person transmission. In addition to the serological investigation 22 , recent analysis of single-cell transcriptomics elucidated the high abundance of cellular receptor ACE2 expression in intestine, suggesting an alternative route for intestinal route of 2019-nCoV entry/shedding in the digestive system. We believe that our results might close in part (if not all) a gap of clinical repertoire of COVID-19 pandemic. More importantly, this finding underscores the urgency and importance of additional concerns with cryptic/asymptomatic/mild cold-like syndromes in the earlier recognition of COVID-19 cases, decreasing the miss diagnosis rate by biased screen and even leading to favorable prognosis by its supportive therapy.

Ethics Statement
This was a retrospective study of COVID-19 conducted in Lishui People's Hospital, Zhejiang Province of China. According to the guideline of the National Health Commission, China, all the confirmed COVID-19 cases received the isolation and supportive therapy in the designated provisional hospital. All the clinical data collection (blood biochemical tests and chest computed tomography) and epidemiological surveys were conducted under the approval by Ethics Committee of Lishui People's Hospital (LLW-FO-401). Given the importance of clinical data sharing in public and academic societies, the routine written informed consent of patients was waived during this special period of ongoing COVID-19 epidemic in China and beyond.

Real-Time Quantitative PCR (qPCR)
The samples of nasal and throat swabs collected from the suspected patients were subjected to viral RNA extraction with QIAamp RNA Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. In this multiplex RT-PCR kit (SMSJ-HX-321B, BioGerm, Shanghai, China), the three target genes of 2019-nCoV referred to ORF-1a/b, E, and N, respectively. These genes were detected with three different fluorescence dyes, namely FAM for ORF-1a/b, HEX/VIC for E, and CY5 for N. In general, the standard curve of amplification was supposed to be plotted in "S" form. The specimens whose cycle threshold (Ct) was less than 35, tested positive for 2019-nCoV on real-time fluorescent qPCR. Of note, the primers designed here exhibited appreciable specificity, in that no cross-reactions with the other four human CoVs (229E, HKU1, OC43, and NL63) were detected.

Phylogenetic Analysis
The nucleotide sequence of RdRp and spike protein sequence of 2019-nCoV isolates were retrieved from 37 the GISAID website (https://gisaid.org/CoV2020), and the equivalent sequences of SARS-CoV and bat SARS-like CoV were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov). Sequence alignment were conducted with Cluatal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). Maximum likelihood-based phylogenetic trees were generated with both MEGA software and TreeView.

Re-analysis of RBD-ACE2 Complex
Cryo-EM structure of full length spiker protein was reported by Wrapp et al. 23 . The X-ray structure of RBD of 2019-nCoV complexed with its cellular receptor ACE2 was resolved by Wang's research group 24 . The detailed interface between S protein and its partner ACE2 was analyzed using the software of PyMol. The critical binding residues were highlighted, which are implicated into the formation of hydrogen bonds in the context of 2019-nCoV interaction with its ACE2 receptor.

Case Reports
We identified an unusual cluster of COVID-19 infections originating from Lishui City of Zhejiang Province, China. It involved three patients (Patient 1 to Patient 3, Table 1). A familial transmission of 2019-nCoV was recorded from Patient 1 to his father, termed Patient 2 (Fig. 1). To our surprise, Patient 3 presenting mild influenza-like illness ( Table 1), was confirmed to be positive for COVID-19. Retrospectively, Patient 3 experienced a close contact with Patient 1 at a dinner party in Qingyuan County, on Jan 18, 2020 (Fig. S1). Of note, this timepoint was around a week before the onset of dry cough ( Table 1 and Fig. 1).
Familial Transmission of 2019-nCoV from Son to Father (Patient 1 to 2) In brief, Patient 1 is a local resident of 45-year old in Qingyuan County, Lishui City. On Jan 18, 2020, he attended a dinner party (Fig. 1). Two days later, series of severe respiratory symptoms were developed in Patient 1, which referred to fever, dry cough, nasal congestion, etc. ( Table 1). On Jan 23, 2020, three days after the onset of clinical presentation ( Fig. 1), he was admitted to local hospital and received supportive therapy. Similar to those of earlier-reported cases of COVID-19 11,12,21 , chest computed tomography (CT) revealed that a number of ground-glass opacities are distributed in two lungs (from upper lobe to lower lobe) of the patient 1 (Figs 2A-C). Indeed, these pathological changes of lungs are indicatives of viral pneumonia. As anticipated, on Jan 25, throat swabs obtained from Patient 1 tested positive for 2019-nCoV ( Fig. 1), following the nucleic acid assays of real-time reversetranscription polymerase chain reactions (RT-PCR). Meanwhile, medical department of local government hunted for all those suspected persons possessing the history of close contact with Patient 1. It returned two hits, namely his father of 72-year old (Patient 2) and a 46-year old man (Patient 3). Of being noteworthy, this confirmed COVID-19 patient 1 was immediately transferred to the provisional designated hospital for isolation and professional therapy. The condition of Patient 1 worsened seriously on Jan 28, 2020 and was greatly improved with stable vital signs on February 3, 2020. Following the supportive therapy as well as antiviral therapeutics, Patient 1 was recovered from illness, and discharged smoothly from hospital on Feb 12, 2020 ( Fig. 1). As for the infection of Patient 1 with 2019-nCoV, we have no exact evidence supporting this is directly correlated with "animal-to-human intraspecies jump" or "person-to-person transmission". While, it is of possibility that Patient 1 might be infected by 2019-nCoV during the journey of returning from Leqing County, to Qingyuan County on Jan 18, 2020. The reason lied in that he passed by Wenzhou City, a seriously affected area of COVID-19 epidemic we have recognized now. In fact, this city has already been closely monitored, whose traffic was under the strict regulation. Different from that of Patient 1, the clinical onset of Patient 2 (a 72-year old man) was characterized with "chills, chest tightness, myalgia, etc.", which was developed on the second day after his son was hospitalized (Table 1). Thus, he was also admitted immediately to local hospital of Qingyuan County on Jan 25. As expected, certain pathological damages of lungs by viral infection were illustrated with chest CT images of Patient 2 (Fig. 2). Not only did the single ground-glass alteration appear in the right lower lobe of lung ( Fig. 2A), but also multi-focal ground-glass opacities dispersed from middle lobe to lower lobe of left lung (Figs 2B-C). In addition, similar scenarios were seen in the right lobe of lungs (Fig. 2D). On Jan 27, Patient 2 was also verified by real-time RT-PCR positive for COVID-19. A similar repertoire of COVID-19 progression (from severe to mild) replayed on the father of Patient 1, after he was transferred to the designated place of isolation ( Fig. 1). Fortunately, Patient 2 was recovered well and allowed to leave hospital on Feb 19, 2020 ( Fig. 1). Thus, we believed this constitutes a case of familial transmission of COVID-19 from son to father.

Influenza-Like Illness Caused by 2019-nCoV in Patient 3
Soon after recognition of Patient 1 as the confirmed COVID-19 case on Jan 25, a young man of 36-year old (designated Patient 3) was tracked and strictly requested to proceed the stay of home isolation on Jan 26 ( Fig. 1). This was because that Patient 3 had ever been closely exposed to Patient 1, the confirmed 2019-nCoV carrier in Qingyuan County, on Jan 18, 2020. Epidemiological analysis showed that i) Patient 1 auto-drove to return his hometown (Qingyuan Country) from Leqing Country, close to Wenzhou City (a severely affected area by COVID-2019 according to current viewpoint) on Jan 18 (Fig. S1); ii) Patient 3 returned Lishui City from Qingyuan after the dinner party on the same day, and auto-drove from Lishui City to Wenzhou City on January 21, visited Qingyuan County on Jan 24, and arrived in his hometown Lishui City on Jan 26 (Fig. S1). During the journey of leaving for Lishui City on Jan 26, Patient 2 was reached by a phone call from the local administrative staff and formally informed to stay at home because of being a suspected case of COVID-19.
Though that Patient 3 was shocked by such unexpected disclosure at this very moment, he came to recognize his own statue of being discontinuously dry coughing, which was supposed to start on Jan 23, 2020 (Fig. 1). Unlike the father and son (Patient 2 & 1), this young person only gave a little bit mild symptom "dry cough", a typical sign of flu-like syndrome ( Table 1). Even during the whole period from home isolation (Jan 26, 2020), centralized isolation (Jan 27,2020), to hospitalization (on Jan 29, 2020), none of the bellowed clinical symptoms was developed in this patient 3, including fever/chills, headache/dizziness, chest tightness, fatigue, etc. (Table 1). This was generally consistent with the almost normal parameters of blood biochemical tests ( Table 2). Chest CT imaging of Patient 3 elucidated the presence of an inflammatory focus in the middle lobe of right lung (Figs 4A-B), implying viral infection. In addition, the small pulmonary nodule was seen in left lower lobe of lung (Fig. 4C), and the pulmonary emphysema was visualized in left lung (Fig. 4D). On Jan 31, 2020, throat and nasal swabs from Patient 3 were subjected to the molecular diagnosis of real-time RT-PCR. Three specific genes 2019-nCoV targeted in this assay separately referred to ORF1a/b, E and N (Fig. S3A), as recommend by China CDC with minor improvement. As a result, the cycle threshold (Ct) was 30.09 for ORF1a/b (Fig. S3B), 30.17 for E (Fig. S3B), and 29.49 for N (Fig. S3C), respectively. Obviously, Patient 3 tested positive for COVID-19 on real-time RT-PCR assays. Together with clinical descriptions, genetic data allowed us to confidently recognize Patient 3 as a cryptic carrier of COVID-19 (Fig. 1). Moreover, the body temperature of this patient behaved at normal level, varying from 35.8°C to 36.8°C, five days post-hospitalization (Fig. S2). This posed the possibility that 2019-nCoV remains inactive in this patient during the 20 days after the onset of dry cough (Fig. 1). It is unusual, but not without any precedent, in that a rare case of asymptomatic COVID-19 carrier was recently reported in German 20 . Thanks to effective therapy, the lug inflammation of Patient 3 was largely relieved (Figs 5A-B). During 10 days of medical observation since the patient transfer on Feb 1, vital signs of Patient 3 remained stable. Thus, he was discharged smoothly from the designated hospital on Feb 11, 2020 (Fig. 1). Given that viral load of 2019-nCoV in upper respiratory tracts of asymptomatic case was recently found to be comparable to that in severe patient 25 , we believed that the potential transmission of mild COVID-19 cases with/without influenza-like illness cannot be neglected. It seemed a bias that we preferred on persons with severe syndrome in the former screen of COVID-19. Therefore, our discovery could benefit the improvement in screening practices of COVID-19, through extending from symptomatic individuals to asymptomatic persons with close contact of the confirmed 2019-nCoV case.

Clinical and Epidemiological Study
Patient 1 and his father, Patient 2, constituted a family cluster of severe pneumonia associated with COVID-19 (Figs 2 and 3). Although none of clinical presentations was shared by the two patients (Table 1), chest CT scans showed that multiple ground-glass opacities consistently occurred in their two lungs (Figs 2 and 3). Such pathological alterations of lungs were generally consistent with the presence of the deadly virus, 2019-nCoV, in the two patients. Because that the clinical onset (like fever) of Patient 1 on Jan 20, was four days earlier than that of his father (Fig. 1), it was reasonable be believe his son was infectious resource within this familial cluster of COVID-19. Patient 1 disclosed that he passed by Wenzhou City during the journey of returning hometown Qingyuan County from Leqing County on Jan 18 (Fig.  S1). As we knew as of Feb 22, 2020, Wenzhou City was one of severely affected area of Zhejiang Province. This is because that 504 individuals were confirmed to be positive for COVID-19 in this city of Wenzhou alone, whereas the whole province of Zhejiang contained 1204 cases of 2019-nCoV infections. Thus, Patient 1 was anticipated to get infected on the way to home on Jan 18, 2020.
Unlike the father and son (Patients 1 & 2) with severe pneumonia, Patient 3 only gave influenza-like illness (ILI) with a hallmark of dry cough, but without any fever ( Table 1). To the best of our knowledge, it is a first report of ILI case associated with 2019-nCoV infections in Zhejiang Province. Epidemiological surveys allowed us to conclude that this is due to the close contact with Patient 1 at the dinner party held in Qingyuan County, Jan18, 2020 (Fig. S1). Patient 3 reported that the clinical onset of "dry cough" appeared on Jan 23, five days after this dinner party (Fig. 1). Despite that almost nothing was unusual in the blood biochemical tests ( Table 2), a focus of inflammation of right lung was detected by chest CT imaginings (Figs 4A-B). This well explained why Patient 3 clinically presented with "dry cough", rather than "high fever and chest tightness" ( Table 1). Thanks to the recognition of Patient 1 to be positive for COVID-19, Patient 3 with a mild symptom was officially requested to stay at home, then admitted to the designated hospital, and finally diagnosed to as a COVID-19 case (Figs 1 and  S4). The fact that the screening practice of 2019-nCoV gave a higher priory to those patients with severe acute respiratory syndrome suggested biased selection criteria used in an earlier stage of the COVID-19 outbreak. Presumably, this led to the neglection of certain asymptomatic COVID-19 cases. Given the carriage of high viral load in asymptomatic COVID-19 cases, its potential capability of human-to-human transmission posed a risky challenge to public health in China and beyond.
Unlike that SARS-CoV is featuring with quasi-species, genetic heterogeneity is barely seen in the 2019-CoVs circulating in China and beyond thus far. It is of possibility that certain variants of 2019-CoV with low virulence appeared to adapt within human host in near future. Therefore, we anticipated that the causative of current COVID-19 epidemic is ancestral version, rather than a sister when compared with SARS-CoV, an etiological agent of the 2003 SARS outbreak 1, 2, 27 .

Alternative 2019-nCoV Transmission in Intestine
Like those of SARS-CoV and MERS-CoV, the spread of 2019-nCoV proceeded primarily by respiratory droplets of the close contacts. The spiker glycoprotein, a type I viral fusion protein of 2019-nCoV is responsible for its efficient binding to the cellular ACE2 receptor 23 , which is followed by the event of cellular and viral membrane fusion 26 . In general agreement with our recent study of modeling 26 , the x-ray crystal structure of 2019-nCoV RBD/ACE2 complex defined a fine binding interface (Fig. S4A), in which a number of contact residues are highlighted (Figs S4B-C) 24 . Thus, an efficient production of the ACE2 receptor is pre-requisite for active entry of 2019-nCoV into the infected host. As expected, high viral loads were recently detected in nasal and throat swabs from patients with COVID-19, because that the upper respiratory tract is attributed to a primary route of 2019-nCoV dissemination 25 . Of particular being noteworthy, serological data suggested that certain anal swabs are positive for 2019-nCoV 22 , which is similar to a scenario seen with the stool specimen reported by Chen and coauthors 11 . It was of possibility that an alternative route of COVID-19 dissemination/shedding occurs in intestine. While it needed further mechanistic evidence.
To address this unanswered question, an approach of single-cell transcriptomics recently was exploited to systematically compare distinct expression profile of cellular receptor ACE2 in different tissues as well as various cell types 28 . Indeed, the expression of ACE2 is variable in different tissues and organs 28,29 . In addition to the ACE2 receptor 28 , the transmembrane serine protease (TMPRSS) that is responsible for the cleavage of spiker protein into two functional domains (S1 and S2), was also determined to be produced appreciably in intestines 29 . This hints an alternative route of 2019-nCoV dissemination and or shedding in intestine, posing a risk of fecal-to-oral spread. Therefore, we speculated that dynamic expression of ACE2 and TMPRSS can be related to viral pneumonia of 2019-nCoV with different severity. Probably it is due to i) the limited number of susceptible ACE2-producing AT2 cells and/or ii) 2019-nCoV infection stagecorrelated expression of ACE2 in certain AT2 cell types of lungs 28,29 . Together, variable expression of ACE2 in patients determined different susceptibility and severity of respiratory symptoms, which can explain the existence of asymptomatic/mild 2019-nCoV carrier, like flu-like syndrome.

Discussion
As of Feb 23, 2020, totally 17 confirmed 2019-nCoV cases with no deaths were accumulated in Lishui City, Zhejiang Province. 14 of 17 patients have been discharged smoothly from the designated provisional hospital. This is in part (if not all) attributed to the rapid and efficient recognition of asymptomatic COVID-19 cases in the incubation time by local government. It seemed likely that Malayan pangolins act as one of intermediate host for 2019-nCoV circulation. This is because that a number of pangolin coronaviruses are identified to share 90% similarity to that of 2019-nCoV 30 . Obviously, the event of genetic re-arrangement is necessitated prior to the onset of 2019-nCoV spread.
The data we reported here, represented a first example that influenzalike illness is caused by the person-to-person transmission of 2019-nCoV. Also, this cluster case of COVID-19 involved a familial transmission of severe viral pneumonia from son to father. Our findings provided additional insights into full spectrum of COVID-19 in various severity. As an infectious resource in this COVID-19 case, Patient 1 disclosed that he had no history of close contact with "Huanan Seafood Wholesale Wet Market". Thus, it ruled out the possibility of animal-to-human dissemination". By contrast, Patient 1 might get infected with 2019-nCoV on the way to his hometown, because that he passed by Wenzhou City with 504 confirmed COVID-19 cases (Fig. S1). Unfortunately, Patient 2, also presented severe pneumonia, which is analogous to that of his son, Patient 1. This was a severe familial transmission of COVID-19. On the contrary, Patient 3 only gave the mild symptom "dry cough" of ILI after his contact with Patient 1. This suggested the diversity of clinical spectrum with those recipients of 2019-nCoV donated from s severe patient. Meanwhile, we are concerned with whether certain mild case of COVID-19 can spread to those contacts, giving severe syndromes. Given that ACE-2 also functioned as a cellular receptor for 2019-CoV, we speculated that different syndromes might be determined by various level of ACE2 expression in susceptible cell types as well as cell counts within the infected host. Because that the analysis of single-cell transcriptomics elucidated higher level of ACE2 expression in intestine than that of lungs, the paradigm organ infected with 2019-nCoV, we concluded that an alternative pathway for 2019-nCoV might exist in intestine. Similar scenarios have ever been seen in other two related coronaviruses, SARS-CoV 31 and MERS-CoV 31 . Very recently, this prediction was supported by the positive detection of 2019-nCoV in anal swabs 22 .
In conclusion, we described a first example that influenza-like illness is caused by the 2019-nCoV transmission from a patient with heavy pneumonia. It was probable that the cryptic carrier of 2019-nCoV is more risky challenge than those severe cases of COVID-19. Thereafter, this underlined the importance of additional concerns with cryptic/asymptomatic/mild cold-like syndromes in the earlier recognition of COVID-19 cases, decreasing the miss diagnosis rate by biased screen.
The plus symbol "+" denotes the presence of certain symptom, whereas the minus symbol "--" refers to the absence of relevant clinical symptoms.  As for Patient 1 (the son of Patient 2 in Qingyuan County of Zhejiang Province, Fig. S1), the onset of COVID-19 is featuring with numbers of symptoms (esp., fever, Table 1) on Jan 18, 2020. He was in-hospitalized in the provincial designated hospital on Jan 23, and then diagnosed as the confirmed case of 2019-nCoV infection. Unlike the scenario of Patient 1, his father (Patient 2) exhibits the flu-like symptoms (like chills and generalized weakness) on Jan 24, went to hospital on the next day, and then identified as a confirmed case of COVID-19 on Jan 25, 2020.
Patient 3 who attended a dinner party along with Patient 1 in Qingyuan County of Zhejiang Province, on Jan 18 (Fig. S1), exclusively displayed dry cough, a representative sign of influenza-like illness from Jan 23 to Jan 29 ( Table 1). Upon on patient 1 was verified to be positive for 2019-nCoV, the patient 3 was regarded as a suspected case, and immediately informed to stay at home on Jan 26, 2020. On Jan 27, he was transferred to the place of centralized isolation. Because of limited relief of dry cough, Patient 3 was hospitalized in Lishui People's Hospital on Jan 29, confirmed as a COVID-19 case on Jan 31, and then transferred to a designated hospital of this city on Feb 1, 2020. Chest CT scans with multiple sections were routinely performed. ground-glass opacity is showed with a red arrow. The chest CT scan with multiple sections was routinely performed, and the ground-glass opacity is highlighted with the red arrow. The vesicular emphysema is highlighted with a magenta arrow.