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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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... In addition to negative impacts of the SARS-CoV-2 on hospital workload [18], medical clinic organization [19], long-term health [20], small business [21], socioeconomic system [22] and employment [23], the national health care systems have to face the need for thousands of laboratory tests per day [24]. The Veterinary Public Health Institutes, namely Istituti Zooprofilattici Sperimentali (IZS), perform the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by RT-PCR on behalf of the Italian Ministry of Health [24]. ...
... In addition to negative impacts of the SARS-CoV-2 on hospital workload [18], medical clinic organization [19], long-term health [20], small business [21], socioeconomic system [22] and employment [23], the national health care systems have to face the need for thousands of laboratory tests per day [24]. The Veterinary Public Health Institutes, namely Istituti Zooprofilattici Sperimentali (IZS), perform the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by RT-PCR on behalf of the Italian Ministry of Health [24]. In the face of the current COVID-19 crisis, the "National Reference Centre for Whole Genome Sequencing of microbial pathogens: database and bioinformatic analysis" (GENP AT) formally established at the IZS dell'Abruzzo e del Molise (IZSAM) in Teramo (G.U.R.I. 196, August 23, 2017), dedicates its developments to improve analytical workflows of SARS-CoV-2 sequences from routine surveillance activities. ...
... Concerning the samples from the first collection, acquisition of sequencing data implied successively sampling (oropharyngeal swab transport medium or bronchoalveolar lavage), virus inactivation (PrimeStore® MTM, in BSL3 biocontainment laboratory), nucleic acid purification (MagMaxTM CORE from Thermofisher), real-time RT-PCR-based SARS-CoV-2 RNA detection (Taq-ManTM 2019-nCoV Assay Kit v1 or v2 from Thermofisher) [24], RNA reverse transcription through multiplexing PCR (primer scheme nCoV-2019/V1) following the ARTIC protocol (https://artic.network/) [109], cDNA purification (AMPure XP beads, Agencourt), cDNA quantification (Qubit dsDNA HS Assay Kit and Qubit fluorometer 2.0 from Thermofisher or QuantiFluor ONE dsDNA System from Promega and FLUOstar OMEGA from BMG Labtech), NGS library preparation (Illumina DNA Prep kit) and sequencing (2 × 150 bp: MiniSeq or NextSeq500 from Illumina). ...
Article
Full-text available
Background Faced with the ongoing global pandemic of coronavirus disease, the ‘National Reference Centre for Whole Genome Sequencing of microbial pathogens: database and bioinformatic analysis’ (GENPAT) formally established at the ‘Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise’ (IZSAM) in Teramo (Italy) is in charge of the SARS-CoV-2 surveillance at the genomic scale. In a context of SARS-CoV-2 surveillance requiring correct and fast assessment of epidemiological clusters from substantial amount of samples, the present study proposes an analytical workflow for identifying accurately the PANGO lineages of SARS-CoV-2 samples and building of discriminant minimum spanning trees (MST) bypassing the usual time consuming phylogenomic inferences based on multiple sequence alignment (MSA) and substitution model. Results GENPAT constituted two collections of SARS-CoV-2 samples. The first collection consisted of SARS-CoV-2 positive swabs collected by IZSAM from the Abruzzo region (Italy), then sequenced by next generation sequencing (NGS) and analyzed in GENPAT ( n = 1592), while the second collection included samples from several Italian provinces and retrieved from the reference Global Initiative on Sharing All Influenza Data (GISAID) ( n = 17,201). The main results of the present work showed that (i) GENPAT and GISAID detected the same PANGO lineages, (ii) the PANGO lineages B.1.177 (i.e. historical in Italy) and B.1.1.7 (i.e. ‘UK variant’) are major concerns today in several Italian provinces, and the new MST-based method (iii) clusters most of the PANGO lineages together, (iv) with a higher dicriminatory power than PANGO lineages, (v) and faster that the usual phylogenomic methods based on MSA and substitution model. Conclusions The genome sequencing efforts of Italian provinces, combined with a structured national system of NGS data management, provided support for surveillance SARS-CoV-2 in Italy. We propose to build phylogenomic trees of SARS-CoV-2 variants through an accurate, discriminant and fast MST-based method avoiding the typical time consuming steps related to MSA and substitution model-based phylogenomic inference.
... To identify and isolate infected persons, millions of RT-PCR tests are carried out around the world [9]. Hence, COVID-19 not only overloads national health care systems but also seriously challenges diagnostic capacities when hundreds or thousands of tests need to be performed per day per laboratory [2,9,10]. Although a large number of in-house and commercial real-time RT-PCR assays have been developed and compared regarding their diagnostic performance within a very short time frame [11][12][13][14][15][16], the timely and comprehensive diagnostics might be impaired by supply shortage of the PCR chemistry, as the availability of test kits has become a major bottleneck. ...
... Since SARS-CoV-2 became a pandemic in early 2020, this virus is keeping the world in suspense as the control measures affect all areas of life, and the disease itself heavily challenges health care systems and diagnostic capacities. To identify and isolate infected persons, inconceivably large numbers of RT-PCR tests need to be performed [2,9,10]. In comparison to spring and summer (the so-called "first wave"), the number of tests even increased in the northern hemisphere's autumn 2020 and winter 2020/21, when many countries have faced a "second wave", i.e., strongly increasing case numbers [23,24]. ...
Article
Full-text available
Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.
... In addition, veterinarians found themselves in the spotlight as animal health experts when SARS-CoV-2 positive animals were identified (3). They demonstrated their expertise in diagnostic testing and outbreak response (4,5). At the very beginning of the pandemic, we engaged second-year veterinary students in a discussion of how the COVID-19 pandemic may impact veterinarians and veterinary medicine. ...
... Veterinarians have significant expertise in animal coronaviruses (39), which should be leveraged in the scientific response to the SARS-CoV-2 outbreak. Furthermore, veterinarians have been at the forefront of SARS-CoV-2 virology research (40,41), epidemiology (5,42,43), diagnostic testing (4,44), and vaccine development (45,46). Ten of the students (42%) remarked that a team approach, with participants representing various areas of expertise, is necessary to address the ecological, biological, and social aspects of disease outbreaks. ...
Article
Full-text available
COVID-19 has had significant effects on the field of veterinary medicine. Adaptation to pandemic-related and post-pandemic challenges requires engagement from all levels of the professional pipeline, including veterinary college students. Insights gained from this group may inform curriculum design, help the veterinary profession innovate, maximize opportunities for positive change, and avoid negative outcomes. The current study aimed to understand the potential impacts of the COVID-19 pandemic on veterinary medicine, as foreseen by second-year veterinary students in an online discussion during a public health course in the spring of 2020. Twenty-one percent of the 113 students agreed to participate in this qualitative research study. We used an inductive coding process and distilled the student responses into descriptive themes to capture diverse perspectives and understand possible post-pandemic pathways for the veterinary profession. Four themes emerged from the student discussion posts, describing how veterinarians might be affected by the COVID-19 pandemic: (1) economic and social impacts, (2) adapting to challenges, (3) collaborations to improve public health, and (4) disparities and diversity. These themes are a starting point for discussion and innovation as veterinarians plan for the post-pandemic world; further investigation will provide additional guidance for veterinary leaders.
... 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). ...
Article
Full-text available
In late December 2019, the first cases of viral pneumonia caused by an unidentified pathogen were reported in China. Two years later, SARS-CoV-2 was responsible for almost 450 million cases, claiming more than 6 million lives. The COVID-19 pandemic strained the limits of healthcare systems all across the world. Identifying viral RNA through real-time reverse transcription-polymerase chain reaction remains the gold standard in diagnosing SARS-CoV-2 infection. However, equipment cost, availability, and the need for trained personnel limited testing capacity. Through an unprecedented research effort, new diagnostic techniques such as rapid diagnostic testing, isothermal amplification techniques, and next-generation sequencing were developed, enabling accurate and accessible diagnosis. Influenza viruses are responsible for seasonal outbreaks infecting up to a quarter of the human population worldwide. Influenza and SARS-CoV-2 present with flu-like symptoms, making the differential diagnosis challenging solely on clinical presentation. Healthcare systems are likely to be faced with overlapping SARS-CoV-2 and Influenza outbreaks. This review aims to present the similarities and differences of both infections while focusing on the diagnosis. We discuss the clinical presentation of Influenza and SARS-CoV-2 and techniques available for diagnosis. Furthermore, we summarize available data regarding the multiplex diagnostic assay of both viral infections.
... Since the beginning of the pandemic, the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise regions (IZSAM) was appointed by the Italian Ministry of Health to support the molecular diagnosis, genomic characterization and phylogenetic analysis of SARS-CoV-2. In addition, IZSAM has been providing the Local Health Authorities of Abruzzo region for epidemiological support when needed Danzetta et al., 2020;Di Giallonardo et al., 2020;Lorusso et al., 2020). From 20 December 2020 onward, IZSAM started to regularly perform Next Generation Sequencing (NGS) on SARS-CoV-2 PCR-positive swabs collected in Abruzzo since the second half of the month. ...
Article
Full-text available
From 24 December 2020 to 8 February 2021, 163 cases of SARS-CoV-2 Alpha variant were identified in Chieti province, Abruzzo region. Epidemiological information allowed the identification of 14 epi-clusters. With one exception, all the epi-clusters were linked to the town of Guardiagrele: 149 contacts formed the network, two-thirds of which were referred to the family/friends context. Real data were then used to estimate transmission parameters: according to our method, the calculated Re(t) was higher than 2 before the 12 December 2020. Similar values were obtained from other studies considering Alpha variant. However, results should be critically interpreted in light of the epidemiological situation and control measures in place in the studied area at that particular time. Italian sequence data were combined with a random subset of sequences obtained from GISAID database. Genomic analysis showed close similarity between the sequences from Guardiagrele, forming one distinct clade. This would suggest one or limited unspecified introductions from outside to Abruzzo region in early December 2020, which led to the diffusion of Alpha in Guardiagrele and in neighbouring municipalities, with very limited inter-regional mixing.
... Positive PCR amplicons were purified using a QIAquick PCR Purification Kit (Qiagen) and were sent to LGC Genomics (Berlin, Germany) for sequencing. RNA purified from one bovine sample was processed for whole genome sequencing and bioinformatic analysis as previously described (Lorusso et al., 2020;Marcacci et al., 2016). The purified RNA was used for the assessment of sequencing independent single primer amplification protocol (SISPA, (Djikeng et al., 2008)). ...
Article
Full-text available
Influenza D virus (IDV) was first isolated in 2011 in the USA and has since been shown to circulate in cattle, pigs, sheep, wild boar, and camels. In Africa, there is limited data on the epidemiology of IDV and, so, we investigated the presence of IDV among domestic ruminants and wild animals in Namibia by screening nasal swabs using an IDV-specific molecular assay. IDV RNA was detected in bovines (n=2), giraffes (n=2) and wildebeest (n=1). The hemagglutinin-esterase-fusion (HEF) gene from one of the bovine and the wildebeest samples was successfully sequenced as well as the full genome for the second bovine sample. Phylogenetic analysis of the HEF gene positioned that the African virus variants within the D/OK lineage but with a long branch. The African variant had an amino acid diversity of 2.41% and most likely represents a distinct genotype within the lineage. Notably, the polymerase acidic protein gene (PA) was more closely related to a different lineage (D/660), indicative of potential reassortment. This is the first genetic characterization of IDV in Africa and it adds important data to our understanding of the global IDV distribution.
... Thus, the One Health concept is useful in understanding the spread, prevention, and control of COVID-19 [2,9,14]. Animal health professionals such as veterinarians play important roles among the One Health team in controlling the pandemic [15]. During this pandemic, AHPs are regarded as essential workers rendering animal healthcare services to communal inhabitants during compulsory stay indoor policy (lockdowns) [16]. ...
Article
Full-text available
Introduction: the on-going COVID-19 pandemic caused by the SARS-CoV-2 virus has imposed serious public health and economic threats on the entire world population. The SARS-CoV-2 has been identified from both domestic and wild animals constituting a threat to humans since most apparently healthy animals may potentially infect and cause the disease in humans, especially, Animal health professionals (AHPs) who come directly in contact with animals. These professionals such as veterinarians play important roles among the One Health team in controlling the pandemic. This survey was conducted to assess the knowledge, risk perception, and prevention behaviors of AHPs in Nigeria who are important personnel in the control of emerging and re-emerging zoonotic and infectious diseases. Methods: an online-based epidemiological cross-sectional pre-tested questionnaire survey was carried out from April to May 2020. A total of 427 AHPs joined this survey. Descriptive statistics, chi-square test, and binary logistic regression model were used to analyze data collected. Results: the respondents have a mean knowledge score of 7.34 ± 2.05 (from a total possible score of 11) with the majority (66.7%, n = 285) having satisfactory knowledge scores on COVID-19. A high proportion (240/353) of respondents reported bats to be the major wildlife incriminated in the transmission of the COVID-19 virus though other animals reported are tiger, monkey, lion, and pangolin. At least, a quarter 131 (30.7%) of respondents perceived that visiting live animal markets is of high zoonotic risk to the spread of coronavirus. Most respondents reported hands washing and sanitizing after handling animals, and using personal protective equipment when handling suspected animals. At p<0.05; respondents' age, marital status, professional status, and geopolitical zone were significantly linked with satisfactory knowledge. Veterinarians (OR=0.40; 95%CI: 0.22–0.75) were thrice less likely to possess unsatisfactory knowledge of COVID-19 than laboratory technologists. Conclusion: participants in this survey have a satisfactory level of COVID-19 knowledge and good mitigation measures instituted while working at their stations. However, AHPs need more enlightenment about the various zoonotic risk pathway contributing to the transmission of COVID-19.
... China has learned a lesson from the previous H1N1 pandemic that science and technology can play a vital role to control the spread of the virus and mitigate the loss that might occur due to the pandemic and related issues [102]. Apart from that, veterinarians also can play a great role in infectious diseases like SARS, COVID-19, and so on, as these diseases are quite familiar to veterinarians for a long time [103]. The experience they (veterinarians) have gained while dealing with the community health system such as controlling and characterisation of earlier health issues of poultry and livestock including avian flu, foot, bluetongue and mouth diseases, are responsible for huge social and economic losses, is no doubt of great help to lessen the impact of the world crisis. ...
Article
Throughout time, the global tourism industry and economy have been significantly affected by disasters and crises. At present, COVID-19 represents one of these disasters as it has been causing a serious economic downturn with huge implications in tourism. In this review paper, we have analysed more than 100 papers regarding the effect and consequences of a pandemic on tourism and related industries, the economic situation in countries and areas, and mitigation of the loss incurred due to pandemic situations. The article (1) is based on past research on tourism and economy, (2) examines the effects of a pandemic on listed sectors and mitigation processes, and (3) suggests future research and approaches to help progress the field. We have gathered and categorised the literature reviews into several parts. In addition, we have listed the name of authors, journal names, books, websites, and relevant data.
... At the same time, there is space for the discussion of more active surveillance, instead of a passive report to OIE from the countries, promoting the searching of animal cases among the cluster of human cases. SARS-CoV-2/COVID-19 deserve a comprehensive approach from the One Health approach (Dasgupta et al. 2021, Leroy et al. 2020, Lorusso et al. 2020. More integration is still needed to increase our understanding of transmission, risks and consequences of this emerging coronavirus disease. ...
Article
Full-text available
Background: COVID-19 pandemic is essentially a zoonotic disease. In this context, early in 2020, transmission from humans to certain animals began reporting; the number of studies has grown since. Objectives: To estimate the pooled prevalence of SARS-CoV-2 natural infection in animals and to determine differences in prevalence between countries, years, animal types and diagnostic methods (RT-PCR or serological tests). Methods: A systematic literature review with meta-analysis using eight databases. Observational studies were included but analyzed separately. We performed a random-effects model meta-analysis to calculate the pooled prevalence and 95% confidence interval (95%CI) for prevalence studies and case series. Results: After the screening, 65 reports were selected for full-text assessment and included for qualitative and quantitative analyses. A total of 24 reports assessed SARS-CoV-2 infection by RT-PCR, combining a total of 321,785 animals, yielding a pooled prevalence of 12.3% (95%CI 11.6%-13.0%). Also, a total of 17 studies additionally assessed serological response against SARS-CoV-2, including nine by ELISA, four by PRTN, one by MIA, one by immunochromatography (rest, two studies, the method was not specified), combining a total of 5,319 animals, yielding a pooled prevalence of 29.4% (95%CI 22.9%-35.9%). Conclusion: A considerable proportion of animals resulted infected by SARS-CoV-2, ranking minks among the highest value, followed by dogs and cats. Further studies in other animals are required to define the extent and importance of natural infection due to SARS-CoV-2. These findings have multiple implications for public human and animal health. One Health approach in this context is critical for prevention and control.
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Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging worldwide. Here, we report the complete genome sequences of 13 severe acute SARS-CoV-2 strains belonging to lineage B.1.525 (variant η).
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The SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China, and has since impacted a large portion of China and raised major global concern. Herein, we investigated the extent of molecular divergence between SARS-CoV-2 and other related coronaviruses. Although we found only 4% variability in genomic nucleotides between SARS-CoV-2 and a bat SARS-related coronavirus (SARSr-CoV; RaTG13), the difference at neutral sites was 17%, suggesting the divergence between the two viruses is much larger than previously estimated. Our results suggest that the development of new variations in functional sites in the receptor-binding domain (RBD) of the spike seen in SARS-CoV-2 and viruses from pangolin SARSr-CoVs are likely caused by natural selection besides recombination. Population genetic analyses of 103 SARS-CoV-2 genomes indicated that these viruses had two major lineages (designated L and S), that are well defined by two different SNPs that show nearly complete linkage across the viral strains sequenced to date. We found that L lineage was more prevalent than the S lineage within the limited patient samples we examined. The implication of these evolutionary changes on disease etiology remains unclear. These findings strongly underscores the urgent need for further comprehensive studies that combine viral genomic data, with epidemiological studies of coronavirus disease 2019 (COVID-19).
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The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-2. 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 infection. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identifcation of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identifed pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. 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 new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.
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The recent epidemics caused by the novel human coronavirus 2019 (2019-nCoV), now referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is having a great impact not only on the Chinese health care system and economy but mainly on the global perception of the risk and on the change of common habits and life styles. The novel coronavirus (CoV) recognises, with high probability, a zoonotic origin but the role of animals in the SARS-CoV-2 epidemiology is still largely unknown. However, CoVs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face CoV infections in animals, which could represent a model for SARS-CoV-2 infection in humans. In the present paper, we provide an up-to-date review of the literature currently available on animal CoVs, focusing on the molecular mechanisms that are responsible for the emergence of novel CoV strains with different antigenic, biologic and/or pathogenetic features. A full comprehension of the mechanisms driving the evolution of animal CoVs will help better understand the emergence, spreading, and evolution of SARS-CoV-2.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19, which was first reported in Wuhan, China in December, 2019. Despite the tremendous efforts to control the disease, COVID-19 has now spread to over 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. Here, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. We found experimentally that cats are susceptible to airborne infection. Our study provides important insights into the animal models for SARS-CoV-2 and animal management for COVID-19 control.
Preprint
Full-text available
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19, which was first reported in Wuhan, China in December, 2019. Despite the tremendous efforts to control the disease, COVID-19 has now spread to over 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. Here, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but efficiently in ferrets and cats. We found that the virus transmits in cats via respiratory droplets. Our study provides important insights into the animal reservoirs of SARS-CoV-2 and animal management for COVID-19 control.
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Full-text available
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.
Article
The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.