Content uploaded by Alessio Lorusso
Author content
All content in this area was uploaded by Alessio Lorusso on May 07, 2020
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Contents lists available at ScienceDirect
One Health
journal homepage: www.elsevier.com/locate/onehlt
A“One-Health”approach for diagnosis and molecular characterization of
SARS-CoV-2 in Italy
Alessio Lorusso
a,⁎
, Paolo Calistri
a
, Maria Teresa Mercante
a
, Federica Monaco
a
, Ottavio Portanti
a
,
Maurilia Marcacci
a
, Cesare Cammà
a
, Antonio Rinaldi
a
, Iolanda Mangone
a
, Adriano Di Pasquale
a
,
Marino Iommarini
b
, Maria Mattucci
c
, Paolo Fazii
d
, Pierluigi Tarquini
e
, Rinalda Mariani
f
,
Alessandro Grimaldi
g
, Daniela Morelli
a
, Giacomo Migliorati
a
, Giovanni Savini
a
, Silvio Borrello
h
,
Nicola D'Alterio
a
a
Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise “G. Caporale”, Teramo, Italy
b
Ospedale San Liberatore Presidio COVID-19 Atri, Teramo, Italy
c
Direzione Sanitaria ASL, Teramo, Italy
d
Reparto di Microbiologia e Virologia clinica, Ospedale Civile Spirito Santo, Pescara, Italy
e
UOSD Malattie Infettive Ospedale G. Mazzini, Teramo, Italy
f
UOC Malattie Infettive Ospedale SS Filippo e Nicola, Avezzano (L' Aquila), Italy
g
UOC Malattie Infettive Ospedale S. Salvatore, L'Aquila, Italy
h
Direzione Generale della Sanita' Animale e dei Farmaci Veterinari, Ministero della Salute, Roma, Italy
ARTICLE INFO
Keywords:
SARS-CoV-2
COVID-19
Molecular characterization
Next generation sequencing
Mutations
Variants
One health
Veterinarians
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. The Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise is currently testing more than 600
samples per day and performing whole genome sequencing from positive samples. Sequence analysis of these
samples suggested that different viral variants may be circulating in Italy, and so in Abruzzo region. 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.
1. Introduction
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 be still 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 per-
centage of them (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 in-
tensive care unit treatment [3]. ARDS is often fatal [4]. The novel
epidemic, recognized as a public health emergency of international
concern on January 302,020, and acknowledged at a pandemic on
March 112,020, was initially recognized in December 2019 in Wuhan
City, Hubei Province, China, and continues to expand [5].
In Italy, up to the 8th 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
https://doi.org/10.1016/j.onehlt.2020.100135
Received 11 April 2020; Received in revised form 16 April 2020; Accepted 16 April 2020
⁎
Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise “G. Caporale”, Campo Boario, 64100 Teramo, Italy.
E-mail address: a.lorusso@izs.it (A. Lorusso).
One Health 10 (2020) 100135
Available online 19 April 2020
2352-7714/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
T
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 now officially reports 4,642 deaths. Since
the infection was first identified in Codogno (Lombardy region) on
February 21st, 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
allows treatment of sick patients without overloading the capacity of
hospitals. Italy established draconian measures by restricting move-
ment 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 the gene expression of CoV during its discontinuous sub-
genomic replication [6]. Downstream, the 5′-most two-thirds of SARS-
CoV-2 genome comprises the replicase gene, which consists of two
overlapping open reading frames, ORF 1a and 1b translated to produce
two large polyproteins, pp1a and pp1b. Cleavage of the replicase
polyproteins is predicted to result in 16 end-products; nsp1–nsp11 en-
coded in ORF1a and nsp12–16 encoded in ORF1b [7]. Located down-
stream of ORF1b are four ORFs that code for structural proteins (spike
(S), envelope (E), membrane (M) and nucleocapsid (N) proteins) and
additional ORFs coding for accessory genes. As SARS-CoV-1, the S
(through the S1) protein mediates viral attachment to the specific cell
receptor angiotensin-converting enzyme type 2 (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
cats [9].
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 and 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 re-
present a network throughout the entire National territory.
2. Materials and methods
This paper aims at describing the first three weeks of experience
gained by the Istituto Zooprofilattico Sperimentale dell' Abruzzo e del
Molise (IZSAM) in the melieu of COVID-19 crisis in support of the di-
agnostic workflow for SARS-CoV-2 of the Abruzzo region. The first case
of COVID-19 in Abruzzo region was recorded on February 27
th
in a
male patient originating from Lombardy region who arrived in Abruzzo
for tourism several days before the movement restrictions implemented
first in Lombardy region and in other provinces of northern Italy, and
then extended all over the Italian territory.
Samples tested for the presence of SARS-CoV-2 RNA are collected
from the respiratory tract of individuals which are either hospitalized,
or screened as for contact history with infected individuals or in the
framework of the screening programs for workers of the SSN. For the
vast majority, samples of hospitalized individuals originate from hos-
pitals located in different cities of Abruzzo region: Teramo (Ospedale
Civile Giuseppe Mazzini), Atri (Ospedale Civile S. Liberatore), Pescara
(Ospedale Civile Spirito Santo, Pescara), Avezzano (Ospedale Civile SS.
Nicola e Filippo), Sulmona (Ospedale SS Annunziata), Lanciano
(Ospedale Renzetti), L'Aquila (Ospedale Regionale S. Salvatore) and
Castel di Sangro (Ospedale Civile).
The workflow for SARS-CoV-2 RNA detection is composed by two
steps. The first includes virus inactivation (PrimeStore®MTM, in BSL3
biocontainment laboratory) starting from a total volume of 200 μlof
oropharyngeal (OF) swab transport medium (physiological solution) or
bronchoalveolar lavage (BAL) and nucleic acid purification
(MagMaxTM CORE) according to the manufacturer's instructions. The
second consists of RNA detection by the TaqMan
TM
2019-nCoV Assay
Kit v1 (Thermofisher, qPCR) whose results are interpreted following the
manufacturer's instructions. Briefly, this test targets three different
portions of SARS-CoV-2 genome located in the replicase, S and N pro-
tein encoding genes, respectively.
Laboratory activities are not limited to the molecular detection of
SARS-CoV-2 RNA. Selected positive samples showing low threshold
cycle (C
T
) values are regularly further processed by next generation
sequencing (NGS) in order to obtain the whole genome sequence of the
occurring strains. At the time this paper has been written, a total
number of 46 samples were processed by NGS. They were selected
within those collected from patients between the 16th and 23rd of
March 2020.
RNA from these infected samples was treated with TURBO DNase
(Thermo Fisher Scientific, Waltham, MA) at 37 °C for 20 min 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’-GCCGGAGCTCTGCAGATATCTTTTTTTTT
TTTTTTTTTTT-3′[22]. The reaction was incubated at 23 °C for 10 min
and at 50 °C for 50 min. After an inactivation step at 80 °C for 10 min,
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 h
and 75 °C for 10 min. Next, 5 μl of the ds cDNA was added to PCR
master mix containing 1× Q5 Reaction Buffer, Q5 High-Fidelity DNA
Polymerase, dNTPs mix and the primer-tag FR20RV 5’-GCCGGAGCTC
TGCAGATATC-3′[11]. The incubation was performed with the fol-
lowing thermal conditions: 98 °C for 1 min, 40 cycles of 98 °C for 10 s,
65 °C for 30 s and 72 °C for 3 min and a final extension step of 72 °C for
2 min. 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 pre-
pared by using Nextera DNA Flex Library Prep (Illumina Inc., San
A. Lorusso, et al. One Health 10 (2020) 100135
2
Diego, CA) according to the manufacturer's protocol. Deep sequencing
was performed on the MiniSeq (Illumina Inc., San Diego, CA) by the
MiniSeq Mid Output Kit (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 un-
mapped reads were retained for 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.
3. Results
Starting from March 16
th
and up to April 8
th
around 8000 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 7994 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 in the GISAID database [15]; as listed in
Table 2. All obtained sequences in this study showed > 99% of nu-
cleotide (nt) identity with Wuhan-Hu-1 (NC_045512) SARS-CoV-2 re-
ference strain. However, all of them had single nucleotide poly-
morphisms (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 (241C > T) which co-evolved with
3037C > T, 14408C > T, and 23403A > G [16]. While 3037C > T
causes a synonymous mutations in nsp3 (F105F) 14408C > T and
23403A > G cause amino acid mutations in RNA primase (nsp 12,
P323L), and S protein (D614G), respectively. The four co-mutations are
prevalent in viral isolates from Europe. All sequences obtained in this
study, but one, had 27046C (T175 in the coded M protein); one se-
quence from Pescara, which was not deposited with GISAID, had the
mutation 27046C > T (T175M in the M protein). Moreover, 29/45 (12/
16 of those which have been deposited) sequences showed R203K and
G204R in the N protein as for the presence of mutations 28881G > A,
28882G > A, and 28883G > C 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
24
th
with a travel history to Italy Regarding Italian sequences, 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 aged 41 years. Interestingly, a
sequence obtained from a sample collected from the hospital of Atri
(TE7097), which was not deposited with GISAID as for suboptimal
horizontal coverage, did not show D614G in the S protein, typical of
European strains, thus retaining the D614 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
Fig. 1. Temporal distribution of samples tested by results and percentage of positive samples.
Table 1
Age mean values of individuals tested positive and negative for SARS-CoV-2. (p-
Value < .0001, two tails Mann-Whitney Test).
Age (years)
Negative Positive
Mean 50.2 55.6
Median 49.6 56.9
Standard deviation 16.5 20.0
A. Lorusso, et al. One Health 10 (2020) 100135
3
the absence of sequence coverage in those portions of the genome. No
mutations were observed in critical residues of the S1 protein.
Genome analysis suggests that different viral SARS-CoV-2 variants
might be circulating in Italy and so in Abruzzo region.
Although the hallmark characterizing SARS-CoV-2 strains observed
in this study are mainly located in the N protein, there is no evidence of
geographical clustering in the Abruzzo region related to the two N
protein viral variants. Sequences showing R203K and G204R in the N
protein, according to GISAID, were evidenced primarily in northern
Europe, but also recently in different countries including, within the
others, USA, Spain, Greece, Vietnam and South America. As there is a
critical lack of SARS-CoV-2 sequences from northern Italy, speculations
upon the origin of the N protein viral variants can be made once a
clearer picture of the genomic characteristics of the viruses circulating
in Italy is available. In this regard, it would be important to obtain the
sequence information of the early SARS-CoV-2 strains detected in
Abruzzo and northern Italian regions to draw evidenced-based con-
clusions. 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 [17]. 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.
4. Discussion
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 col-
laborations 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, including the current SARS-CoV-2, are zoonotic. Moreover, they
0
50
100
150
200
250
300
Age (years)
Number of tested
Negave Posive
Fig. 2. Number of positive and negative samples by age (years) of patients.
Table 2
SARS-CoV-2 sequenced and deposited with GISAID. M, male; F, female. Age is expressed in years.
Strain GISAID acc.no Hospital Sex, Age N genotype Vertical coverage Horizontal coverage
hCoV-19/Italy/TE4836/2020 EPI_ISL_418260 Teramo M, 41 R203, G204 346 X 98,90%
hCoV-19/Italy/TE4959/2020 EPI_ISL_418259 Pescara M, 76 K203, R204 197 X 99,98%
hCoV-19/Italy/TE5056/2020 EPI_ISL_418257 Teramo F, 75 K203, R204 297 X 99,99%
hCoV-19/Italy/TE4880/2020 EPI_ISL_418256 Atri M, 80 K203, R204 1268 X 99,31%
hCoV-19/Italy/TE4925/2020 EPI_ISL_418255 Pescara F, 63 K203, R204 1751 X 99,70%
hCoV-19/Italy/TE4953/2020 EPI_ISL_418258 Pescara M, 87 K203, R204 2462 X 99,98%
hCoV-19/Italy/TE5052/2020 EPI_ISL_418261 Teramo F, 78 R203, G204 87 X 95,19%
hCoV-19/Italy/TE5166/2020 EPI_ISL_420563 Teramo M, 68 R203, G204 2501 X 100%
hCoV-19/Italy/TE5472/2020 EPI_ISL_420564 Castel di Sangro M, 54 R203, G204 1960 X 99,98%
hCoV-19/Italy/TE5476/2020 EPI_ISL_420565 Teramo M, 61 K203, R204 224 X 99,85%
hCoV-19/Italy/TE5512/2020 EPI_ISL_420566 L' Aquila M, 71 K203, R204 101 X 99,67%
hCoV-19/Italy/6193/2020 EPI_ISL_420568 Teramo M, 43 K203, R204 3721 X 99,87%
hCoV-19/Italy/TE6225/2020 EPI_ISL_420592 Teramo F, 29 K203, R204 126 X 98,85%
hCoV-19/Italy/TE5780/2020 EPI_ISL_420567 L'Aquila M, 64 K203, R204 447 X 99,86%
hCoV-19/Italy/TE6195/2020 EPI_ISL_420569 Teramo M, 86 K203, R204 2850 X 99,97%
hCoV-19/Italy/TE6222/2020 EPI_ISL_420583 Teramo M, 38 K203, R204 538 X 99,94%
A. Lorusso, et al. One Health 10 (2020) 100135
4
are also equipped with large infrastructure for genomic analysis and
storage of sequence data. These infrastructures are routinely used in
animal health and food security emergences and for diagnostic pur-
poses. In this regard, the analysis of the whole viral genome of SARS-
CoV-2 strains is a critical task. However, still scarce is genomic data
(and related metadata) available from SARS-CoV-2 strains circulating in
Italy and further efforts are necessarily warranted. Whole genome se-
quencing straight from infected biological samples may indeed provide
useful information to identify mutations during the virus adaptation to
humans, such as mutations in critical residues of the S protein or re-
sulting in the loss of accessory genes as already described for SARS-
CoV-1 [18]. 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 veter-
inarians 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, and 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 char-
acterization, 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 [5,19], 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 profes-
sionals 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 es-
tablishment 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
viruses are SARS-rCoVs identified from pangolins [23], however, the
exact origin of SARS-CoV-2 has yet to be demonstrated. In this per-
spective, veterinary virologists may surely support this important task
as well as those doomed to understand 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 [20].
As for cats, recent evidences also demonstrated that they might get
infected from COVID-19 positive humans (https://www.nature.com/
articles/d41586-020-00984-8) or following experimental infection
[21], thus confirming the high affinity of SARS-CoV-2 with feline ACE2.
Although the role of domestic animals in the epidemiology of SARS-
CoV-2 seems to be negligible, further studies are reasonably warranted.
Moreover, to plan future strategies for SARS-CoV-2 containment, it will
be essential to better understand the protective role of the various
classes of antibodies against the virus, as well as the prevalence of
serologically positive individuals in the human population when the
epidemic curve has shown a stable decrease. Also for these purposes,
the IZSs' laboratories will be useful for processing a large number of
serum samples and to support the Public Health Institutes in the ne-
cessary experimental studies.
Declaration of Competing Interest
Authors declare no conflict of interest.
Acknowledgments
The authors deeply acknowledge all the health care workers in-
cluding doctors, nurses, technicians, medical staff, administrators, food
and cleaning service workers, pharmacists, and all members of the
COVID-19 diagnostic group at IZSAM. Mention of trade names or
commercial products in this article is solely for the purpose of providing
specific information and does not imply recommendation or endorse-
ment by the IZSAM. Funding were provided by the SSN.
References
[1] P. Zhou, X.L. Yang, X.G. Wang, B. Hu, L. Zhang, W. Zhang, H.R. Si, Y. Zhu, B. Li,
C.L. Huang, H.D. Chen, J. Chen, Y. Luo, H. Guo, R.D. Jiang, M.Q. Liu, Y. Chen,
X.R. Shen, X. Wang, X.S. Zheng, K. Zhao, Q.J. Chen, F. Deng, L.L. Liu, B. Yan,
F.X. Zhan, Y.Y. Wang, G.F. Xiao, Z.L. Shi, A pneumonia outbreak associated with a
new coronavirus of probable bat origin, Nature (2020), https://doi.org/10.1038/
s41586-020-2012-7.
[2] A.E. Gorbalenya, S.C. Baker, R.S. Baric, R.J. de Groot, C. Drosten, A.A. Gulyaeva,
B.L. Haagmans, C. Lauber, A.M. Leontovich, B.W. Neuman, D. Penzar, S. Perlman,
L.L.M. Poon, D.V. Samborskiy, I.A. Sidorov, I. Sola, J. Ziebuhr, The species Severe
acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming
it SARS-CoV-2, Nat. Microbiol. 4 (2020) 536–544, https://doi.org/10.1038/
s41564-020-0695-z (Epub 2020 Mar 2).
[3] X. Yang, Y. Yu, J. Xu, H. Shu, J. Xia, H. Liu, et al., Clinical course and outcomes of
critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-cen-
tered, retrospective, observational study, Lancet Respir. Med. (2020), https://doi.
org/10.1016/S2213-2600(20)30079-5 Epub 2020/02/28.
[4] M.A. Matthay, R.L. Zemans, G.A. Zimmerman, et al., Acute respiratory distress
syndrome, Nat. Rev. Dis. Primers 5 (2019), https://doi.org/10.1038/s41572-019-
0069-0.
[5] A. Lorusso, P. Calistri, A. Petrini, G. Savini, N. Decaro, Novel coronavirus (SARS-
CoV-2) epidemic: a veterinary perspective, Vet. Ital. (2020), https://doi.org/10.
12834/VetIt.2173.11599.1.
[6] T. Li, Y. Zhang, L. Fu, C. Yu, X. Li, Y. Li, X. Zhang, Z. Rong, Y. Wang, H. Ning, et al.,
siRNA targeting the leader sequence of SARS-CoV inhibits virus replication, Gene
Ther. 12 (2005) 751–761.
[7] J. Ziebuhr, E.J. Snijder, A.E. Gorbalenya, Virus-encoded proteinases and proteolytic
processing in the Nidovirales, J. Gen. Virol. 81 (2000) 853–879.
[8] A.C. Walls, Y.J. Park, M.A. Tortorici, A. Wall, A.T. McGuire, D. Veesler, Structure,
function, and antigenicity of the SARS-CoV-2 spike glycoprotein, Cell (2020),
https://doi.org/10.1016/j.cell.2020.02.058 pii: S0092–8674(20)30262–2.
[9] K.G. Andersen, A. Rambaut, W.I. Lipkin, et al., The proximal origin of SARS-CoV-2,
Nat. Med. (2020), https://doi.org/10.1038/s41591-020-0820-9.
[10] M. Marcacci, E. De Luca, G. Zaccaria, M. Di Tommaso, I. Mangone, G. Aste,
G. Savini, A. Boari, A. Lorusso, Genome characterization of feline morbillivirus from
Italy, J. Virol. Methods 234 (2016) 160–163.
[11] T. Allander, M.T. Tammi, M. Eriksson, A. Bjerkner, A. Tiveljung-Lindell,
B. Andersson, Cloning of a human parvovirus by molecular screening of respiratory
tract samples, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 12891–12896.
[12] A.M. Bolger, M. Lohse, B. Usadel, Trimmomatic: a flexible trimmer for illumina
sequence data, Bioinformatics 30 (2014) 2114–2120, https://doi.org/10.1093/
bioinformatics/btu170.
[13] B. Langmead, S. Salzberg, Fast gapped-read alignment with bowtie 2, Nat. Methods
9 (2012) 357–359.
[14] H. Li, B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth,
G. Abecasis, R. Durbin, 1000 Genome Project Data Processing Subgroup, The
Sequence alignment/map (SAM) format and SAMtools, Bioinformatics 25 (2009)
2078–2079.
[15] Y. Shu, J. McCauley, GISAID: Global initiative on sharing all influenza data –from
vision to reality, EuroSurveillance 22 (2017), https://doi.org/10.2807/1560-7917.
ES.2017.22.13.30494.
[16] C. Yin, Genotyping Coronavirus SARS-CoV-2: Methods and implications.
arXiv:2003.10965 [q-bio.GN], 2020.
[17] P. Zhao, J. Cao, L. Zhao, Z. Qin, J. Ke, W. Pan, H. Ren, J. Yu, Z. Qi, Immune re-
sponses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine,
Virology 331 (2005) 128–135.
[18] J. Cui, F. Li, Z.L. Shi, Origin and evolution of pathogenic coronaviruses, Nat. Rev.
Microbiol. 17 (2019) 181–192.
[19] N. Decaro, A. Lorusso, Novel human coronavirus (SARS-CoV-2): a lesson from an-
imal coronaviruses, Vet. Microbiol. (2020), https://doi.org/10.1016/j.vetmic.2020.
108693.
[20] N. Decaro, V. Martella, L.J. Saif, C. Buonavoglia, COVID-19 from veterinary med-
icine and one health perspectives: what animal coronaviruses have taught us, Res.
Vet. Sci. 131 (2020) 21–23.
[21] J. Shi, Z. Wen, G. Zhong, H. Yang, C. Wang, R. Liu, X. He, L. Shuai, Z. Sun, Y. Zhao,
A. Lorusso, et al. One Health 10 (2020) 100135
5
L. Liang, P. Cui, J. Wang, X. Zhang, Y. Guan, H. Chen, Z. Bu, Susceptibility of ferrets,
cats, dogs, and other domesticated animals to SARS-coronavirus 2, Science (2020),
https://doi.org/10.1126/science.abb7015 In press.
[22] A. Djikeng, R. Halpin, R. Kuzmickas, J. Depasse, J. Feldblyum, N. Sengamalay,
C. Afonso, X. Zhang, N.G. Anderson, E. Ghedin, D.J. Spiro, Viral genome sequencing
by random priming methods, BMC Genomics 9 (5) (2008), https://doi.org/10.
1186/1471-2164-9-5.
[23] T.T. Lam, M.H. Shum, H.C. Zhu, Y.G. Tong, X.B. Ni, Y.S. Liao, W. Wei, Y.W. Cheung,
W.J. Li, L.F. Li, G.M. Leung, E.C. Holmes, Y.L. Hu, Y. Guan, Identifying SARS-CoV-2
related coronaviruses in Malayan pangolins, Nature (2020), https://doi.org/10.
1038/s41586-020-2169-0.
A. Lorusso, et al. One Health 10 (2020) 100135
6
