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1 Epidemiological description and analysis of RdRp, E and N genes dynamic

2 by RT-PCR of SARS-CoV-2 in Moroccan population: Experience of the
3 National Reference Laboratory (LNR)-UM6SS

4 Benrahma houda1,2* , Diawara Idrissa2,3, Smyej Imane2, Rahoui Jalila 2, Meskaouni Nida2,
5 Benmessaoud Rachid2, Arouro Khadija2, Jaras Khadija2, Moujid Fatima Zahra 2, Adam
6 Zahra2, Nahir Salma2 , Aouzal Zineb2,Elguezzar Hajar2, Jeddane Leila2, Ousti Fadoua2, EL
7 Bakkouri Jalila2,4, Nejjari Chakib5

9 1 - Faculty of Medicine, Mohammed VI University of Health Sciences (UM6SS), Casablanca,

10 Morocco.

11 2 -National Reference Laboratory, Mohammed VI University of Health Sciences (UM6SS),

12 Casablanca, Morocco.

13 3- Faculty of Nursing and Allied Health Sciences, Mohammed VI University of Health Sciences
14 (UM6SS), Casablanca Morocco

15 4-Laboratory of Clinical Immunology, Inflammation and Allergy (LICIA), Faculty of Medicine and
16 Pharmacy, Hassan II University, Casablanca, Morocco.

17 5-International School of Public Health, Mohammed VI University of Health Sciences (UM6SS),

18 Casablanca, Morocco.

19 * Corresponding author: [email protected]

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30 Abstract:

31 The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome
32 coronavirus 2 (SARS-CoV-2), is a new infectious disease that first emerged in Hubei
33 province, China, in December 2019. On 2 March 2020, the Moroccan Ministry of Health
34 confirmed the first COVID-19 case in Morocco. The new virus SARS-CoV-2 was identified
35 in the sample of a Moroccan expatriate residing in Italy. Without a therapeutic vaccine or
36 specific antiviral drugs, early detection and isolation become essential against novel
37 Coronavirus.

38 This study aims to analyze the epidemiological profile of the SARS-CoV-2 in Moroccan cases
39 and to investigate the dynamic of RdRp, N, and E genes in patients from diagnosis until the
40 recovery.

41 Among 859 COVID-19 RT-PCR tests realized for 376 patients, 187 cases had positive results
42 COVID-19. 4% were positive with the 3 genes RdRp, N, and E, 40 % with N and E genes, 3%
43 with RdRp and N genes, 31% with only the RdRp gene and 22% cases are positives
44 with N gene. The analysis of the Covid-19 genes (RdRp, N, and E) dynamic reveal that more
45 than 6% stay positive with detection of the N and E gene, and 14% with the N gene after 12
46 days of treatment.

47 The median period from positive to the first negative Covid-19 RT-PCR tests was 6.8±2.24
48 days for 44% cases, 14.31± 2.4 days for 30%, and 22.67 ± 1.21 days for 4%.

49 This a first description of the Moroccan COVID-19 cases and the analysis of the dynamic of
50 the RdRp, N, and E genes. The analysis of our population can help to improve in the care of
51 patients.

52

53 Keywords: COVID-19, SARS-CoV-2, RdRp gene, N gene, E gene, RT-PCR, Morocco.

54

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60 1. Introduction

61 According to the World Health Organization (WHO), the WHO China Country Office was
62 informed of cases of pneumonia of unknown etiology in Wuhan City, Hubei Province, on
63 December 31, 2019 [1].

64 The investigations identified a new virus that was closely related to severe acute respiratory
65 syndrome coronavirus (SARS -CoV) [2].The novel coronavirus in humans was initially
66 named as 2019-nCoV [3], and then designated as SARS-CoV-2 by the Coronavirus Study
67 Group of the International Committee on Taxonomy of Viruses [4]. And WHO announced the
68 epidemic disease caused by SARS-CoV-2 as coronavirus disease 2019 (COVID-19) [5].

69 On April 28th, 2020, 213 countries and territories included Morocco, with more than 2
70 959 929 confirmed cases and more than 202 733 have died from the rapidly-spreading SARS-
71 CoV-2 virus [6].

72 On the 2sd of March 2020, the Ministry of Health confirmed the first COVID-19 case in
73 Morocco. The virus was detected in a Moroccan expatriate residing in Italy and who came
74 from Italy on February 27th, 2020. Also, the second case was confirmed by the end of the
75 same day, involving an 89-year-old woman Moroccan residing in Italy too who had returned
76 to Morocco on the 25th of February 2020. Till the 28th of April 2020, 4,246 confirmed cases of
77 COVID-19 with 163 deaths [7].

78 Among the foremost priorities to facilitate public health interventions in Morocco is a reliable
79 laboratory diagnosis. For that, the Ministry of Health expands screening tests across the
80 country as part of the country’s preparation to end confinement measurement and to rapidly
81 evaluate more possible cases. On April 1st, 2020, the National Reference Laboratory (LNR) of
82 Mohammed VI University of Health Sciences (UM6SS), became the fourth laboratory in
83 Morocco authorized to carry out the biological screening and diagnostics of COVID-19.

84 In acute respiratory infection, RT-PCR is routinely used to detect causative viruses from
85 respiratory secretions. For ensuring the diagnostic of Covid-19, the LNR has been equipped
86 with different platforms to performed real-time RT-PCR testing for three targets in the virus:
87 the envelope (E), the RNA dependent RNA polymerase (RdRp) and the nucleocapsid (N).

88 The coronavirus SARS-CoV-2 genome consists of a leader sequence, ORF1ab encoding

89 proteins for RNA replication, and genes for non-structural proteins (nps) and structural
90 proteins [8]. Like other betacoronaviruses, the SARS-CoV-2 genome encodes four major
 medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135137.this version posted June 20, 2020. The copyright holder for this preprint
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91 structural proteins. The structural proteins are involved in various viral processes, including
92 virus particle formation. The structural proteins include spike (S), envelope (E), membrane
93 protein (M), and nucleoprotein (N), which are common to all coronaviruses [9, 10].

94 To date, no studies exploring the variation of RdRp, N and E genes expression of SARS-CoV-
95 2 in the patient’s specimen. In this study, in first time, we analyses the epidemiological profile
96 of the SARS-CoV-2 in Moroccan cases and in second time we explored the dynamic of RdRp
97 , N and E genes in patients from diagnosis until the recovery.

98

99 2. Material and Methods

100 2.1.Patients and samples

101 All samples included in this study were routinely tested for the presence of SARS-CoV-2
102 immediately upon arrival in the National Reference Laboratory (LNR) from the Cheikh
103 Khalifa International University Hospital, Mohammed VI University of Health Sciences
104 (UM6SS). In this study, we included a total of 376 patients admitted to hospital between
105 March 28, 2020, and April 29, 2020.

106 Nasopharyngeal swab samples were collected for extracting SARS-CoV-2 RNA from patients
107 suspected of having COVID-19 infection. The collected swabs were placed into the transport
108 tube where includes a universal transporting medium that is room temperature stable (UTM-
109 RT).

110 The LNR provide a RT-PCR to clinically suspected COVID-19 patients when they were
111 admitted. Multiple repeat RT-PCR tests were performed during admission in the hospital in a
112 different period based on national mandatory management guidelines.

113 2.2.RNA extraction

114 The RNA extraction from nasopharyngeal samples was performed using AccuPrep ® Viral
115 RNA Extraction Kit (Bioneer Corporation, Korea) according to the manufacturer's
116 instructions. Briefly, swabs were vortexed for 10 second speed followed by extraction from
117 200 µl of UTM-RT, and all samples were subjected to extraction with an elution volume 50
118 μl.

119

120
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121

122 2.3.RT-PCR SARS-CoV2 detection

123 One-Step Reverse Transcription Real-Time polymerase chain reaction (RT-PCR) was used to
124 confirm the presence of the SARS-CoV-2 genome by amplification of RdRp, E, and N gene.
125 For the samples included in this study, we realized the RT-PCR by using GeneFinderTM
126 COVID-19 Plus RealAmp Kit according to the manufacturer's protocol (OSANG Healthcare
127 Co., Ltd, South Korea).

128 The RT-PCR assays were performed on the CFX96 Touch Real-Time PCR Detection System
129 (Bio-Rad Laboratories, Inc.). The reaction mixture contains 10 μl of COVID-19 PLUS
130 Reaction Mixture, 5μl of COVID-19 PLUS Probe Mixture, and 5μl of sample RNA. Total
131 Reaction volume is 20μl per sample.

132 The RT-PCR assays were performed under the following conditions: reverse transcriptional
133 reaction at 50 °C for 20 minutes, pre- denaturation at 95 °C for 5 minutes, 45 cycles of
134 denaturation at 95 °C for 15 seconds and extending and collecting fluorescence signal at 58
135 °C for 60 seconds.

136 The result interpretation was performed according to the manufacturer's protocol (table 1)

137 Table 1: Results interpretation of GeneFinderTM COVID-19 Plus RealAmp Kit

Ct range
Results Of RT-PCR
RdRp E N IC

≤ 43 ≤ 43 ≤ 43 ≤ 35

COVID-19 Positive ≤ 43 ≤ 43 U.D ≤ 35

≤ 43 U.D ≤ 43 ≤ 35

COVID-19 Positive if RdRp ≤ 43 after the second test ≤ 43 U.D U.D ≤ 35

COVID-19 Positive if E and N ≤ 43 after the second U.D ≤ 43 ≤ 43 ≤ 35

test

COVID-19 Positive if N ≤ 43 after the second test U.D U.D ≤ 43 ≤ 35

Beta coronavirus U.D ≤ 43 U.D ≤ 35

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Negative U.D U.D U.D ≤ 35

138 U.D= undetermined, Ct: Cycle Threshold, IC: Internal Control

139 2.4.Statistical analysis

140 Continuous variables were presented as means ± standard deviation (SD). For categorical
141 variables were presented as counts and percentages. P-values less than 0.05 were considered
142 as statistically significant. All statistical analyses were performed using STATA software,
143 version 11.0.

144 3. Results
145 3.1.General characteristics

146 In this study, the total number of Covid-19 RT-PCR assays realized on the 376 included
147 COVID-19 patients was 859, with 3.7 tests per patient. The mean age was 46.82 ± 20.54 years
148 old, comprising 55.76 % men and 44.24% women (table 2).

149 Of the 376 patients, 189 (50.3 %) had negative results and 187 (49.7%) had positive results
150 for Covid-19 RT-PCR tests. In the negative cases, the men present 53.16%, and the women
151 46.84% (table 2).

152 In the Covid-19 positives cases, the gender distribution is 41.83% women and 58.14% men.

153 Among the Covid-19 positive patients, we identify 129 sporadic and 58 family cases (with
154 known exposure history). Family with SARS-CoV-2 infection corresponds to 24 families.
155 The analysis of these families showed that 71% included 2 cases, 21% included 3 cases, 4%
156 included 4 cases, and 4% included 5 cases,

157 3.2.SARS-CoV-2 Genes dynamic

158 The interpretation of the RT-PCR results for the first diagnosis of the three genes RdRp, N,
159 and E showed that: 4% of cases are positive with the 3 genes RdRp, N, and E. 31% cases are
160 positive with only the RdRp gene, 3% of cases are positive with RdRp and N gene, 22% cases
161 are positive with N gene, and 40 % with N and E gene. (tab2)

162 The analysis of the Covid-19 genes RdRp, N, and E dynamic revealed that more than 6% of
163 the SARS-CoV-2 infected person stay positive with detection of the N and E gene, and 14%
164 of the positive patients stay positive with the N gene after 12 days the treatment. After more
165 than 21 days of treatment, the RT-PCR test reveals the persistence of the N gene in 4 cases
166 (3%).
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167 From symptoms and positive Covid-19 RT-PCR test to the first negative Covid-19 RT-PCR
168 tests, the median period was 6.8±2.24 days for 44% cases.

169 For 30% cases the median period 14.31± 2.4 days for. A longer period was identified in 4% of
170 cases with a median period of 22.67 ± 1.21 days. For 22% of cases the median period was
171 2.03 ± 0.88 days, for these patients the RT-PCR was realized for the first time by other
172 reference laboratories (tab2). For all patients with the first negative test for the 3 genes, a
173 second RT-PCR test was realized after 24h (table 2).

174 We also investigate the impact of age on the profile of Covid-19 by RT-PCR. The Test of
175 homogeneity is carried out by comparing the positive and negative cases for a different range
176 of age. A significant association was observed for the over than 64 years old range with p =
177 0.009 and OR= 1.74, 95% CI= [1.28, 2.33].

178

179 Table 2: clinical characteristics of all patients, and gene dynamic in positives cases

Variable All patients

Clinical parameters
Gender (%)
Men 55.76
Women 44.24
Age (mean ± SD) years 46.82 ± 20.54
SARS-CoV-2 RT-PCR assay
Total tests 859
Tests/patient 3.75 /patient
Positive tests 187
Sporadic positive cases 129
Families positive cases 58
Onset of symptom to negative test, median period
(days)
For 44% cases 6.8±2.24 days
For 30% cases 14.31± 2.4 days
For 22% cases 2.03 ± 0.88 days
For 4% cases 22.67± 1.21 days
Gene dynamic after days treatment (133 cases) Positives cases %
After 12 days
Presence N and E gene 16
Presence N gene 14
After 21
N gene 3
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180

181

182 4. Discussion

183 The current SARS-CoV-2 outbreak is the third epidemic attributed to coronavirus in the 21st
184 century, and incredibly the number of confirmed SARS-CoV-2 infection has surpassed SARS
185 and MERS in world wild [1, 11].

186 This is the first scale report from 285 COVID-19 Moroccan patients with 859 samples of RT-
187 PCR tests for Covid-19 detection. The data of this study is the preliminary analysis of the
188 Moroccan population analyzed in the LNR which will allow making more in-depth studies of
189 patients infected by the SARS-CoV-2.

190 After analysis of the cases implicated in our study, we found that the infection was prone to
191 affect mem more than women with different percentages of 55.76% and 44.24% respectively.
192 This result consistent with many conclusions from different studies [12, 13].

193 Many studies suggested that coronavirus infected older more than young individuals [14]. In
194 our study, comparing positive and negative cases for a different range of age we found a
195 significate difference (p = 0.009 and OR= 1.74, 95% CI= [1.28, 2.33]), for the range age ≥ 64
196 years. Various studies confirm our observation which suggests that older individuals are more
197 likely to be infected by the virus [12, 15]. We can explain the correlation between age and
198 virus by the presence of higher levels of angiotensin converting enzyme 2 in older people
199 alveoli which are thought to be a receptor for SARS [16].

200 This study included 129 sporadic and 58 familial cases. Various studies analyses the
201 mechanism of transmission of SARS-CoV-2, and they confirmed that the Human to Human
202 transmission via droplets is the main route of transmission within a susceptible population.
203 But no rule out transmission by asymptomatic carriers [17]. The first cases in Morocco were
204 traveled from the epidemic region in Italy. And family members who traveled from Europe
205 were most likely responsible for a familial cluster of COVID-19 once back home. Another
206 explication of the presence of clusters in our country is the organization of familial activity
207 (wedding, death ceremonies, etc…), and some industrial activity.

208 The SARS-Cov-2 share similar sequencing characteristics with SARS-CoV and MERS-CoV,
209 but the analysis of different case series showed that the shedding pattern of the viral nucleic
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210 acid of patients infected with SARS-CoV-2 is different from SARS-CoV. In the early stage,
211 the SARS-CoV had a modest viral load peaked approximately 10 days after symptoms onset.

212 For the SARS-CoV-2, the median duration of the virus in the respiratory sample was 18 days,
213 and from symptom onset, the peak viral shedding in respiratory specimens of positive cases
214 occurred after about 10 to days for the SARS-CoV [18, 19]. In our study, we found that the
215 duration of virus shedding in lower respiratory tract samples was longer than 14 days for 30%
216 of cases, and peak viral shedding occurred after about two weeks from symptom onset.
217 Tracing the duration of the virus is very important for effective control and prevention of the
218 epidemic.

219 Because highly sensitive and specific diagnostic assays are the key to the identification of
220 cases, contact tracing, for diagnosis of cases with suspected SARS-CoV-2 infection. We
221 chose to performed detection of unique viral sequences with RT-PCR by using
222 GeneFinderTM COVID-19 Plus RealAmp Kit (OSANG Healthcare Co., Ltd, South Korea),
223 which is one of the validated protocols for in vitro diagnostic (CE marked) available on the
224 market. The RT-PCR assays target the E gene encoding for the envelope protein, which is
225 common to the Sarbecovirus subgenus, the second specific assay targets the RdRp gene
226 encoding for RNA-dependent RNA polymerase and the third assay target the N gene encoding
227 nucleocapsid protein [20, 21].

228 Based on the RT-PCR tests of positive cases during hospitalization period, we found that after
229 12 days of treatment, more than 6% of the SARS-CoV-2 infected person stay positive with
230 the detection of the N and E gene, and 14% of the positive patients stay positive with
231 the N gene. And only 3% stay positive with the N gene after more than 21 days of treatment.

232 To our knowledge, the dynamic of the three SARS-CoV-2 genes was not analyzed before.
233 The genome SARS-CoV-2 shares similar sequencing characteristics with SARS-CoV and
234 MERSCoV. Human coronaviruses are positive-sense RNA (30 kb) viruses. Two types of
235 proteins characterize Human coronavirus, structural (Spike (S), Nucleocapsid (N), Matrix
236 (M), and Envelope (E)) and non-structural proteins (nsp1 up to nsp16) including the RNA
237 dependent RNA polymerase (RdRp) (nsp12). The organization of the coronavirus genome is
238 5′-leader-UTR- replicase-S (Spike)-E (Envelope)-M (Membrane)- N (Nucleocapsid)-3′ UTR-
239 poly (A) tail [22].

240 If we analyze the Coronavirus life cycle, we found that the initial attachment of the virion to
241 the host cell is initiated by interactions between the S protein and its receptor. Following
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242 receptor binding, the virus must next gain access to the host cell cytosol. This is generally
243 accomplished by acid dependent proteolytic cleavage of S protein [23]. The RdRp is a vital
244 enzyme for the life cycle of RNA viruses, because the ability of coronavirus to recombine is
245 tied to the strand switching ability of this protein. Recombination likely plays a prominent
246 role in viral evolution [23]. For the nucleocapsid (N) protein, the primary function is to
247 package the viral RNA genome within the viral envelope into a ribonucleoprotein (RNP)
248 complex called the capsid. Ribonucleocapsid packaging is a fundamental part of viral self-
249 assembly and replication. Additionally, the N-protein of the SARS-CoV-2 affects host cell
250 responses and may serve regulatory roles during its viral life cycle [24].

251 In our study, we found that the N gene persists in cases sample with Ct ≤ 40 in RT-PCR. To
252 explain this persistence, different studies showed that the coronavirus N protein is abundantly
253 produced within infected cells. And these proteins have multiple functions, including binding
254 to viral RNA to form the ribonucleocapsid and have also been proposed to have roles in virus
255 replication, transcription, and translation [24]. Also, in Humans cells, N proteins have been
256 shown to cause deregulation of the cell-cycle, inhibit the production of interferon and induce
257 apoptosis in serum deprived cells, of all which may have possible pathological consequences
258 [24, 25].

259

260 5. Conclusion

261 In summary, this is the first description of the dynamic of RdRp, N, and E genes of the SARS-
262 CoV-2 virus among COVID-19 positive patients. The study allow us to focus on several
263 parameters involved in the care of patients We highlighted that the comprehension of
264 dynamism of these tree genes is an important parameter in the COVID -19 diagnosis. We plan
265 to include in the second time the clinical parameters and the history of infection, to explain
266 this dynamic of this infection. A comprehensive understanding of COVID-19 will help to
267 control the disease.

268

269 Data Availability

270 All data underlying the results are available as part of the article, and no additional source
271 data are required.

272
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273 Declaration of Competing Interest

274 All authors declare that there are no conflicts of interest.

275

276

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