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A “One-Health” approach for diagnosis and molecular characterization of SARS-CoV-2 in Italy

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  • IZS Istituto Zooprofilattico Sperimentale Teramo

Abstract and Figures

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.
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One Health
journal homepage: www.elsevier.com/locate/onehlt
AOne-Healthapproach 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 Zooprolattico 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 conrmed cases and 17,669 deaths have been notied, 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 Zooprolattici
Sperimentali (IZSs), Veterinary Public Health Institutes, in the diagnosis of SARS-CoV-2 by testing human
samples. The Istituto Zooprolattico 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 dierent 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 u, 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 conrmed
cases and 17,669 deaths have been conrmed, 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 Zooprolattico 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 ocially reports 4,642 deaths. Since
the infection was rst identied 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 atten the curveof 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; nsp1nsp11 en-
coded in ORF1a and nsp1216 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 specic 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 S1S2 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 anity to ACE2 also from ferrets and
cats [9].
The WHO denes a conrmed case as a person with laboratory
conrmation 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 Zooprolattici 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 rst three weeks of experience
gained by the Istituto Zooprolattico Sperimentale dell' Abruzzo e del
Molise (IZSAM) in the melieu of COVID-19 crisis in support of the di-
agnostic workow for SARS-CoV-2 of the Abruzzo region. The rst 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
rst 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 dierent 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 workow for SARS-CoV-2 RNA detection is composed by two
steps. The rst 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 purication
(MagMaxTM CORE) according to the manufacturer's instructions. The
second consists of RNA detection by the TaqMan
TM
2019-nCoV Assay
Kit v1 (Thermosher, qPCR) whose results are interpreted following the
manufacturer's instructions. Briey, this test targets three dierent
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 Scientic, Waltham, MA) at 37 °C for 20 min and then
puried by RNA Clean and Concentrator-5 Kit (Zymo Research). RNA
was used for the assessment of sequencing independent single primer
amplication protocol (SISPA) with some modication [10]. Briey,
cDNA was obtained by reverse-transcription (RT) using SuperScript®IV
Reverse Transcriptase (Thermo Fisher Scientic, Waltham, MA) and a
combination of two primers including the random-tagged primer
FR26RV-N 5-GCCGGAGCTCTGCAGATATCNNNNNN-3with 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 (35exo-) (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 Buer, 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 nal extension step of 72 °C for
2 min. The PCR product was puried by ExpinTM PCR SV (GeneAll
Biotechnology CO., LTD Seoul, Korea) and then quantied 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 rst week of testing, not more than
150200 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 rst 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/epiu-applications/next-hcov-19-app/), these
mutations in the N protein rst 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 identied 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 dierent 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 dierent 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 scientic 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
Negave Posive
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 eorts 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 inuenced the choice
of appointing IZSs to support the SSN's eort 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 nding 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 u, bluetongue, foot and mouth disease, and BSE,
which were responsible for huge economic losses. This aptitude of being
ready to actionduring 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 dierent profes-
sionals operating within the SSN is crucial to properly and eectively
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 anis, identied as strain BatCoVRaTG13 from a
faecal sample in Yunnan province, China; and that the next closest
viruses are SARS-rCoVs identied 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 hyperinammation 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 conrming the high anity 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 conict of interest.
Acknowledgments
The authors deeply acknowledge all the health care workers in-
cluding doctors, nurses, technicians, medical sta, 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
specic information and does not imply recommendation or endorse-
ment by the IZSAM. Funding were provided by the SSN.
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... COVID-19 was first reported in December 2019 in humans in connection with the Huanan seafood wholesale market where various species of farmed and wild animals are usually sold (Wuhan, Hubei Province, China) [5][6][7][8]. Since 31 December 2019 and as of week 2022-22, 531,470,423, cases of COVID-19 (in accordance with the applied case definitions and testing strategies in the affected countries) were reported, including 6,318,391 deaths [9]. In the absence of effective drugs and a vaccine, in a fully susceptible population, from the starting of the epidemic in less than one year, SARS-CoV-2 resulted in >30 million confirmed cases (2 million in Europe) of infection worldwide and over 900,000 deaths (185,000 in Europe) [10,11]. ...
... From 31 December 2019, >290,000 confirmed cases of SARS-CoV-2 infection and >35,000 deaths were reported in nine months [13]. Besides harmful impacts on workload and organization of hospitals [14] and medical clinics [15], as well as on health [16] and employment [17][18][19] of populations, the SSN had to perform thousands of daily tests [7,8]. Lockdown and other emergency measures, such as restriction on mobility, social distancing and the closure of all non essential services, were applied. ...
... RNA purified was processed by means of several methods. The first included a metagenomics approach by the combination of the sequence-independent single-primer amplification (SISPA) with Nextera DNA Flex Library Prep (Illumina Inc., San Diego, CA, USA) [8]. The second protocol provided for a targeted approach by the enrichment of some SISPA libraries using myBaits Expert Virus-SARS-CoV-2 kit (Arbor Biosciences, Ann Arbor, MI, USA). ...
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The Istituti Zooprofilattici Sperimentali (IZSs) are public health institutes dealing with the aetiology and path-ogenesis of infectious diseases of domestic and wild animals. During Coronavirus Disease 2019 epidemic, the Italian Ministry of Health appointed the IZSs to carry out diagnostic tests for the detection of SARS-CoV-2 in human samples. In particular, the IZS of Abruzzo and Molise (IZS-Teramo) was involved in the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by Real Time RT-PCR. Activities and infrastructures were reor-ganised to the new priorities, in a "One Health" framework, based on interdisciplinary, laboratory promptness, accreditation of the test for the detection of the RNA of SARS-CoV-2 in human samples, and management of confidentiality of sensitive data. The laboratory information system-SILAB-was implemented with a One Health module for managing data of human origin, with tools for the automatic registration of information improving the quality of the data. Moreover, the "National Reference Centre for Whole Genome Sequencing of microbial pathogens-database and bioinformatics analysis"-GENPAT-formally established at the IZS-Teramo, developed bioinformatics workflows and IT dashboard with ad hoc surveillance tools to support the metagenomics-based SARS-CoV-2 surveillance, providing molecular sequencing analysis to quickly intercept the variants circulating in the area. This manuscript describes the One Health system developed by adapting and integrating both SILAB and GENPAT tools for supporting surveillance during COVID-19 epidemic in the Abruzzo region, southern Italy. The developed dashboard permits the health authorities to observe the SARS-CoV-2 spread in the region, and by combining spatio-temporal information with metagenomics provides early evidence for the identification of emerging space-time clusters of variants at the municipality level. The implementation of the One Health module was designed to be easily modelled and adapted for the management of other diseases and future hypothetical events of pandemic nature.
... Los paquetes diagnósticos que emplean RT-PCR para la detección del SARS-CoV-2 funcionan mediante la lectura de la ARN polimerasa dependiente del ARN (RdRp), fragmentos ORF1ab, el gen de la envoltura (gen E), el gen de la proteína nucleocápside (gen N) (13,16,17,18,19,20) y el gen S (11,16) . Con el fin de mejorar la sensibilidad de la detección, la mayoría de los fabricantes eligen dos o más regiones diana de la secuencia de ácido nucleico viral (11,13,21) . ...
... Los paquetes diagnósticos que emplean RT-PCR para la detección del SARS-CoV-2 funcionan mediante la lectura de la ARN polimerasa dependiente del ARN (RdRp), fragmentos ORF1ab, el gen de la envoltura (gen E), el gen de la proteína nucleocápside (gen N) (13,16,17,18,19,20) y el gen S (11,16) . Con el fin de mejorar la sensibilidad de la detección, la mayoría de los fabricantes eligen dos o más regiones diana de la secuencia de ácido nucleico viral (11,13,21) . ...
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Introducción: la reacción en cadena de la polimerasa con transcriptasa inversa es una técnica de alta precisión en la detección y amplificación de material genético. Objetivo: describir las bases del empleo de la reacción en cadena de la polimerasa con transcriptasa inversa como prueba diagnóstica en la detección del SARS-CoV-2. Método: se realizó una revisión de la literatura en artículos publicados hasta mayo de 2020. Se consultaron las bases de datos: Scopus, Wiley Online Library, SciELO, DIALNET, EBSCO, MEDLINE y PubMed. Se recuperaron artículos en español e inglés, seleccionándose 43 referencias. Desarrollo: la reacción en cadena de la polimerasa con transcriptasa inversa para detectar SARS-CoV-2 consiste en la lectura de la ARN polimerasa dependiente del ARN, fragmentos ORF1ab, el gen E, el gen N y el gen S. El exudado nasofaríngeo ofrece mejores resultados que el orofaríngeo y saliva como muestra. Resulta necesaria la inclusión de pruebas reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) que utilicen especímenes de hisopado rectal en casos sospechosos falsos negativos. Nuevos estudios y técnicas se elaboran con el objetivo de optimizar el proceso de detección. Conclusiones: la disponibilidad de pruebas diagnósticas es crucial para el aislamiento de casos positivos y el seguimiento de la cadena epidemiológica de transmisión. La RT-PCR resultó ser la prueba de elección durante el período de replicación viral. La Prueba de amplificación isotérmica mediada por bucle (RT-LAMP) es una alternativa diagnóstica rápida con principios similares a la RT-PCR.
... This test makes it possible to determine, by taking a nasal sample, if a person is infected. Reverse transcription (RT)-PCR test is a test that takes a few seconds and can be more or less painful depending on individual sensitivity [14]. To confirm or deny the presence of the virus, deep nasal cells are taken using a swab. ...
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Background: This work is a report of the present situation and management of Corona Virus (CoV) infection in the Principality of Monaco. Methods: To comply with the practices adopted by the World Health Organization (WHO) and neighboring Countries in this area, residents infected by CoV are the object of this health report. The Prince’s Government also communicates the data relating to screening. These are stopped at Sunday January 1st included. Results: The results of this survey point out how this infection is still active in Monaco, although not at the emergency levels of 2020. Conclusions: These results call for not leaving prevention measures, both primary (vaccine) and secondary (personal protective equipment [PPE], social distancing, etc.) adopted until now and which gave so good results.
... After Turbo DNAse (Termo Fisher Scientifc, Waltham, MA, USA) incubation, and RNA purifcation by RNA Clean & Concentrator ™ -5 Kit (Zymo Research, Irvine, CA, USA), purifed RNA was processed using a modifed sequenceindependent single-primer-amplifcation (SISPA) method to obtain dsDNA [28,29]. ...
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Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.
... However, the daily use of next generation sequencing (NGS)-based techniques in the HRSV milieu is limited. Indeed, the current COVID-19 pandemic highlighted once more the importance of NGS-based techniques as a fundamental tool helping scientists to study the SARS-CoV-2 mutations over time and genomic epidemiology [10,11]. Until recently, no whole genome sequence data for HRSV was available for infections in Tunisia. ...
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Human orthopneumovirus (HRSV) is a virus belonging to the Pneumovirus genus that causes lower respiratory tract infections (LRTI) in infants worldwide. In Tunisia, thousands of infants hospitalized for LRTI are found to be positive for HRSV but no whole genome sequences of HRSV strains circulating in this country are available thus far. In this study, five nasal swab samples collected at different time points from a three-month-old female baby with severe immunodeficiency that was hospitalized for acute bronchiolitis were investigated by next generation sequencing. The Tunisian sequences from this study originated from samples collected in 2021, belong to the ON1 genotype of HRSV-A, and are clustered with European sequences from 2019 and not from 2020 or 2021. This is most likely related to local region-specific transmission of different HRSV-A variants due to the COVID-19 related travel restrictions. Overall, this is the first report describing the whole genome sequence of HRSV from Tunisia. However, more sequence data is needed to better understand the genetic diversity and transmission dynamic of HRSV.
... Meanwhile, Lu et al. generated 53 genomes from infected patients in the Guangdong region and demonstrated that the infections were likely to be related to travel and not local communities . Similarly, Lorruso et al. used NGS to trace infections from the Abruzzo region to a sequence originating in a sample from Northern Europe with a travel history to Italy based on the presence of the R203K and G204R mutations in the N protein (Lorusso et al., 2020). ...
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The ongoing outbreak of viral pneumonia in China and beyond is associated with a novel coronavirus, SARS-CoV-2¹. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection². Although bats are likely reservoir hosts for SARS-CoV-2, the identity of any intermediate host that might have facilitated transfer to humans is unknown. Here, we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity to SARS-CoV-2 in the receptor-binding domain. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of novel coronaviruses and should be removed from wet markets to prevent zoonotic transmission.
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