key: cord-1016753-7897zxdm
authors: Lorusso, Alessio; Calistri, Paolo; Mercante, Maria Teresa; Monaco, Federica; Portanti, Ottavio; Marcacci, Maurilia; Cammà, Cesare; Rinaldi, Antonio; Mangone, Iolanda; Di Pasquale, Adriano; Iommarini, Marino; Mattucci, Maria; Fazii, Paolo; Tarquini, Pierluigi; Mariani, Rinalda; Grimaldi, Alessandro; Morelli, Daniela; Migliorati, Giacomo; Savini, Giovanni; Borrello, Silvio; D'Alterio, Nicola
title: A “One-Health” approach for diagnosis and molecular characterization of SARS-CoV-2 in Italy
date: 2020-04-19
journal: One Health
DOI: 10.1016/j.onehlt.2020.100135
sha: 9f9a13f950c0c174bed9b2ec493408fa9910cb10
doc_id: 1016753
cord_uid: 7897zxdm

Abstract The current pandemic is caused by a novel coronavirus (CoV) called SARS-CoV-2 (species Severe acute respiratory syndrome-related coronavirus, subgenus Sarbecovirus, genus Betacoronavirus, family Coronaviridae). In Italy, up to the 2nd of April 2020, overall 139,422 confirmed cases and 17,669 deaths have been notified, while 26,491 people have recovered. Besides the overloading of hospitals, another issue to face was the capacity to perform thousands of tests per day. In this perspective, to support the National Health Care System and to minimize the impact of this rapidly spreading virus, the Italian Ministry of Health involved the Istituti Zooprofilattici Sperimentali (IZSs), Veterinary Public Health Institutes, in the diagnosis of SARS-CoV-2 by testing human samples. IZSAM, is currently testing more than 600 samples per day and WGS from positive samples. Sequence analysis of these samples suggested that outbreaks in Abruzzo region may be related to outbreaks of northern Italy and northern Europe, these latter as for the presence of mutations in the N protein. CoVs, and related diseases, are well known to veterinarians since decades. The experience that veterinarians operating within the Public health system gained in the control and characterization of previous health issues of livestock and poultry including avian flu, bluetongue, foot and mouth disease, responsible for huge economic losses, is certainly of great help to minimize the impact of this global crisis.

The current pandemic caused by a novel coronavirus (CoV) called SARS-CoV-2 has been named by the World Health [1,2] Organization (WHO) as COVID- 19 . Even if 80% of COVID-19 human cases are mild, they can still be distressing and long-lasting. Most common symptoms of the infection are fever, dry cough, and shortness of breath. About 20% of infected patients may develop severe cases, and a small percentage of them (5%) about 5% may become critically ill. Patients with severe cases usually develop pneumonia or acute respiratory distress syndrome (ARDS), a condition that may require mechanical ventilation and intensive care unit treatment [3] . ARDS is often fatal [4] . The novel epidemic, recognized as a public health emergency of international concern on January 30 2020, and acknowledged at a pandemic on March 11 2020, was initially recognized in December 2019 in Wuhan City, Hubei Province, China, and continues to [5] .

In Italy, up to the 8 th of April 2020, overall 139,422 confirmed cases and 17,669 deaths have been confirmed, while 26,491 people have recovered (data source: National Department of Italian Civil Protection, available at: http://arcg.is/C1unv). Italian policy makers continue to urge people to stay at home and observe social distancing. Italy is experiencing more deaths than China, the country where the infection originated, which continues to report 3,281 deaths. Since the infection was first identified in Codogno (Lombardy region) on February 21 st , in less than three weeks, COVID-19 overloaded the National Health Care System (Servizio Sanitario Nazionale, SSN) in the northern Italy. It turned the hard hit Lombardy region into a grim glimpse of what countries may expect if they cannot slow down the spread of the virus and ''flatten the curve'' of new cases, which in turn allow treatment of sick patients without overloading the capacity of hospitals. Italy established draconian measures by restricting movement and closing all stores except for pharmacies, groceries and other social essential services. However, these measures did not come in time to prevent the surge of cases that has deeply taxed the capacity even of a well -regarded health care system. SARS-CoV-2 belongs to the species Severe acute respiratory syndrome-related CoVs (SARS-rCoV) within the subgenus Sarbecovirus, genus Betacoronavirus together with SARS-CoV-1 strains from humans and SARS-rCoVs from wild carnivores and horseshoe bats (genus Rhinolophus) [2] .

The virus harbors a linear single-stranded positive RNA genome of nearly 30 kb. At the very 5'-end of the genome is a leader sequence which is the unique characteristic in CoV replication and plays critical roles in for accessory genes. As SARS-CoV-1, the S (through the S1) protein mediates viral attachment to specific cell receptor ACE2 [1] and fusion between the envelope and plasma membrane. As for other CoVs, the S protein is also the main inducer of virus-neutralising antibodies. The S protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site at the S1-S2 boundary through the insertion of 12 nucleotides, which additionally lead to the predicted acquisition of three O-linked glycans around the site [8] . Six residues of the receptor binding domain (RBD) have been shown to be critical for binding to ACE2 receptors and for determining the host range of SARS-CoV-1 like viruses. Based on structural studies and biochemical experiments, SARS-CoV-2 seems to have an RBD that binds with high affinity to ACE2 also from ferrets and

The WHO defines a confirmed case as "a person with laboratory confirmation of COVID-19 infection irrespective of clinical signs and symptoms". Indeed, another issue to face, in the eye of the storm, was the capacity to perform thousands of tests per day. It is reasonable to understand that reliable an d fast diagnosis of COVID-19 infection is a critical task to be performed. Without accurate collection of data and metadata on COVID-19 spread we cannot possibly understand how the pandemic is progressing. In this perspective, to support the SSN and to minimize the impact of this rapidly spreading virus, the Italian Ministry of Health (MoH) involved the Istituti Zooprofilattici Sperimentali (IZSs) in the diagnosis of SARS -CoV-2 by testing human samples. IZSs are Public Health institutes which are coordinated by the MoH and act as technical and operative support of the National Health Care System with regard to animal health, healthiness and quality control for foods of animal origin, breeding hygiene and correct relation between human and animal settlements and the environment. They are ten and represent a network throughout the entire National territory.

This paper aims at describing the first three weeks of experience gained by the Istituto Zooprofilattico at 37°C for 20 minutes and then purified by RNA Clean and Concentrator-5 Kit (Zymo Research). RNA was used for the assessment of sequencing independent single primer amplification protocol (SISPA) with some modification [10] . Briefly, cDNA was obtained by reverse-transcription (RT) using SuperScript® IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) and a combination of two primers including the random-tagged primer FR26RV-N 5'-GCCGGAGCTCTGCAGATATCNNNNNN-3' with a poly-A tagged primer FR40RV-T 5'-GCCGGAGCTCTGCAGATATCTTTTTTTTTTTTTTTTTTTT-3' [11] . The reaction was incubated at 23°C for 10 minutes and at 50°C for 50 minutes. After an inactivation step at 80°C for 10 minutes, 2.5 units of Klenow Fragment (3'→5' exo-) (New England Biolabs, Ipswich, MA) was directly added to the reaction to perform the second strand cDNA synthesis. The incubation was carried out at 37°C for 1 hour and 75°C for 10 minutes. Next, 5 µl of the ds cDNA was added to PCR master mix containing 1X Q5 Reaction Buffer, Q5

High-Fidelity DNA Polymerase, dNTPs mix and the primer-tag FR20RV 5'-GCCGGAGCTCTGCAGATATC-3' [11] .

The incubation was performed with the following thermal conditions: 98°C for 1 minute, 40 cycles of 98°C for 10 seconds, 65°C for 30 seconds and 72°C for 3 minutes and a final extension step of 72°C for 2 minutes.

The PCR product was purified by ExpinTM PCR SV (GeneAll Biotechnology CO., LTD Seoul, Korea) and then quantified using the QuantiFluor One ds DNA System kit (Promega). Libraries were prepared by using (300-cycles) and standard 150 bp paired-end reads. Reads obtained were trimmed by trimmomatic [12] and mapped on the host genome (GCF_000001405) using bowtie2 [13] ; only unmapped reads were retained for J o u r n a l P r e -p r o o f Journal Pre-proof downstream analysis. SARS-CoV-2 consensus sequence was obtained using samtools suite [14] after reads was mapped to reference sequence (NC_045512, Wuhan-Hu-1) by bowtie2.

Starting from March 16 th and up to April 8 th around 8,000 samples were processed at IZSAM. In the first week of testing, not more than 150-200 samples per day were tested, but in the following days the laboratory capacity was increased up to around 600 samples/day. Overall, 839 out of 7,994 samples tested positive by qPCR (Fig 1) . Correlation between qPCR-negative/positive samples and age is showed in Table 1 and Fig 2. Out of 46 samples sent for NGS, 45/46 sequences were suitable for downstream analysis. Only one sequence was discarded as only few reads were obtained. Out of 45 sequences, 16 were complete or almost complete (horizontal coverage >95,2%) and with high vertical coverage. They were deposited with the GISAID database [15] ; as listed in Table 2 . All obtained sequences in this study showed >99% of nucleotide (nt) identity with Wuhan-Hu-1 (NC_045512) SARS-CoV-2 reference strain. However, all of them had SNPs with respect the reference Wuhan-Hu-1 sequence. All sequences either partial or complete, show a first common SNP mutation in the leader sequence (C241T) which is co-evolved with C3037T, C14408T, and A23403G resulting in amino acid mutations in nsp3 (synonymous mutation), RNA primase (P323L), and S protein (D614G), respectively. According to a recent published study, these three co-mutations are in critical proteins for RNA replication (C241T, C14408T) and the S protein (A23403G) for binding to ACE2 receptor [16] . These four co-mutations are prevalent in viral isolates from Europe. Strains from Abruzzo show 28144T in the ORF8 protein sequence, thus they have a leucin residue at position 84 in that accessory protein, and 8782C (S75 in nsp4); thus, these strains may be classified as S type [17] . All sequences obtained in this study, but one, had 27046C (T175 in the coded M protein); one sequence from Pescara, which was not deposited with GISAID, had the mutation C27046T (T175M in the M protein). Moreover, 29/45 (12/16 of those which have been deposited) sequences showed R203K and G204R in the N prote in as for the presence of mutations G28881A, G28882A, and G28883C in the nucleotide sequence. For 3/45 of sequences, the obtained sequence reads did not cover that portion of genome. According to GISAID (Genomic epidemiology of hCoV, https://www.gisaid.org/epiflu-applications/next-hcov-19-app/), these mutations in the N protein first appeared in a SARS-CoV-2 sequence from northern Europe (hCoV-19/Netherlands/Berlicum_1363564/2020, EPI_ISL_413565) originating from a sample collected on February 24th. The same mutations were also identified in one sequence recently released from the Laboratory of Virology Lazzaro Spallanzani (Rome) and collected on February 28 th from a male patient of 41 years old.

Interestingly, a sequence obtained from a sample collected from the hospital of Atri (TE7097), which was not deposited with GISAID as for sub optimal horizontal coverage, did not show D614G in the S protein, J o u r n a l P r e -p r o o f typical of European strains, thus retaining the 614D of early Chinese strains. Unfortunately, we could not investigate for the presence of D614G co-mutations and residues in position 203 and 204 of the N protein as for the absence of sequence coverage in those portions of the genome. No mutations were observed in critical residues of the S1 protein.

Genome analysis may suggest, with all due caution, that SARS-CoV-2 was introduced in Abruzzo region through, mainly two unrelated routes, the first likely related to the COVID-19 outbreak in the northern Italy characterized by SARS-CoV-2 strains with R203 and G204 in the N protein and the other connected to cases detected first in northern Europe characterized by K203 and R204 and by the same constellation of mutations (with respect to the reference genome) described in the replicase and S proteins.

So far, there is no evidence of geographical clustering in the Abruzzo region related to the two N Protein viral variants. However, as there is a critical lack of SARS-CoV-2 sequences from northern Italy, these preliminary considerations should be re-evaluated once a clearer picture of the genomic characteristics of the viruses circulating in Italy will be available. Furthermore, it would be also important to obtain the sequence information of the early SARS-CoV-2 strains detected in Abruzzo to draw robust conclusions. So far, sequences showing R203K and G204R in the N protein, according to GISAID, were detected primarily in northern Europe, but also recently in different countries including, within the others, USA, Spain, Greece, Vietnam and South America. The N protein of SARS-CoV-1 is responsible for the formation of the helical nucleocapsid during virion assembly. The N protein may cause an immune response and has potential value in vaccine development [18] . Hence, these mutations shall be considered when developing a vaccine using the N protein. Reasonably, the role of these mutations needs to be investigated by proper biochemical and reverse genetics experiments.

Diagnosis of SARS-CoV-2 is currently performed in Italy and so in Abruzzo region, in a One Health perspective, with the support of the network of the IZSs. This decision arose by the combination of various relevant factors. Firstly, the IZSs belong to the SSN, coordinated by the MoH, and such condition facilitates the establishment of fruitful collaborations with the Public Health sectors, including the development of common diagnostic and data exchange protocols. Secondly, each IZS has the technical and scientific capacities to support the SSN to meet the extraordinary surge in demand for diagnostic testing of human samples for SARS-CoV-2. Lastly, IZSs have also experience in quality assurance, biosafety, biosecurity, and high throughput testing for the surveillance and control of infectious diseases in animals, some of which, humans, such as mutations in critical residues of the S protein or resulting in the loss of accessory genes as already described for SARS-CoV-1 [19] . An additional factor which may have influenced the choice of appointing IZSs to support the SSN's effort against COVID-19 was related to the biological nature of the occurring agent. CoVs act as primary actors within the so-called human/animal interface across which a plethora of infectious pathogens has been observed to emerge, spill over various species and eventually evolve, thus finding new ecological niches and causing new epidemiological phenomena. Of value, in the Italian context, is certainly the experience that veterinarians operating within the Public health system gained in the control and characterization of previous health issues of livestock and poultry including avian flu, bluetongue, foot and mouth disease, BSE which were responsible for huge economic losses. This aptitude of being "ready to action" during a health emergence certainly includes rapid diagnosis, epidemiological investigations, molecular/antigenic characterization, development of vaccines, and planning of surveillance programs, a process that is pursued, together with saving patients' lives in hospitals, by technicians and scientists around the globe for COVID-19. We add to this the fact that veterinarians have known of CoVs and related diseases for decades [5, 20] , thus, the One Health concept is central and should again be sublimated and adopted to control critical health emergencies, including that of antimicrobial resistance. Therefore, the multidisciplinary involvement of different professionals operating within the SSN is crucial to properly and effectively face the challenges posed by viruses like SARS-CoV-2. A holistic and One Health approach is the sole solution for better understanding the epidemiological aspect of this disease and possibly preventing the establishment of new transmission chains. Currently, the available genome sequences so far clearly reveal that the most closely related virus (96.2% of nt sequence identity) to SARS -CoV-2 is a strain from a bat, Rhinolophus affinis, identified as strain BatCoVRaTG13 from a faecal sample in Yunnan province, China; and that the next closest virus are SARS-rCoVs identified from pangolins [21] , however, the exact origin of SARS-CoV-2 has yet to be demonstrated. In this perspective, veterinary virologists may surely support this important task as well as those doomed to understan d SARS-CoV-2 virulence factors through the assessment of reverse genetics studies and animal models, and to analyze the impact of the hyperinflammation observed in COVID-19 infected patients, characterized by a cytokine storm. This latter evidence is not novel for veterinarians as it is observed in cats infected with feline infectious peritonitis virus, a lethal pathotype of the feline enteric coronavirus [22] .

As for cats, recent evidences also demonstrated that they might get infected from COVID-19 infected humans (https://www.nature.com/articles/d41586-020-00984-8) or following experimental infection [23] diagnostic group at IZSAM. Mention of trade names or commercial products in this article is solely for th e purpose of providing specific information and does not imply recommendation or endorsement by the IZSAM. Funding were provided by the SSN. 

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The authors deeply acknowledge all the health care workers including doctors, nurses, technicians, medical staff, administrators, food and cleaning service workers, pharmacists, and all members of the COVID-19