Coronavirus, a genus of
the Coronaviridae family, is an enveloped virus with a large
plus-strand RNA genome [1].
Human coronavirus (HCoV) 229E and OC43 are
members of group I and II, respectively. HCoV infection is
thought to be responsible for up to 30% of common cold cases in
winter and occasionally causes acute lower respiratory tract
disease in susceptible infants, elderly individuals, and
immunocompromized adults [2-3]. In 2003, the outbreak of severe
acute respiratory syndrome (SARS) led to the preliminary
identification of SARS- coronavirus, [4] which is currently
considered to be a distinct member of the group 2 coronaviruses
[5] or the first
member of group 4 coronaviruses. In 2004, a new HCoV-NL63, was
identified in clinical specimens from both infants and adults
with acute respiratory tract infection (ARTI) in the Netherlands
[1]. Sequence analysis of the complete genome of HCoV-NL63
revealed that the virus was more closely related to HCoV-229E
than to the other human coronaviruses [1]. Preliminary data
suggested that HCoV-NL63 might be an important respiratory tract
pathogen in children, similarly to respiratory syncytial virus
(RSV) and human metapneumovirus (hMPV) [1,6-9]. The epidemilogy
and clinical features of HCoV-NL63 infection are largely
unknown. We prospectively detected the presence of HCoV-NL63 in
children with acute lower respiratory tract infections (ALRTI)
for two consecutive years.
Methods
All children hospitalized with a diagnosis of
ALRTI, between April 2006 to March 2008 were enrolled.
Nasopharyngeal aspirates (NPA) were collected following informed
consent, on the day of admission according to a standard
protocol and transported to the virology diagnostic laboratory
within 4h. The supernatants and cells of NPA were separated by
centrifugation [10] and the cell-free fluid was stored in
aliquots at -80ºC until RNA extraction. Total RNA was
extracted by using the QIAamp viral RNA mini kit (Qiagen,
Germany) and complementary DNA (cDNA) was synthesized by using
the PrimeScript RT Reagent Kit (TaKaRa, China). Primers used for
HCoV-NL63 gene amplification have been previously described
[1,11]. The bulk PCR products were purified using a QIAquick Gel
Extraction kit (Qiagen, Germany). Sequence analyses of the PCR
products were performed on an ABI 3100 sequencer at core
facility of nucleotide sequencing of Chongqing Medical
University. The sequenced fragments of the HCoV-NL63 1a gene
were assembled and analyzed with the SEQMAN, EDITSEQ, and
MEGALIGN programs in the Lasergene program (DNASTAR, Madison,
WI). Phylogenetic trees were generated by the neighbor-joining
method using the MEGA4 program.
Co-infection with other viruses. RSV and
hMPV in specimens were detected by traditional PCR and Real-time
PCR, respectively. Positive samples for HCoV-NL63 were also
subjected to direct immunofluorescence (DFA) (Diagnostic
Hybrids, America) testing for common respiratory viruses,
including influenza A and B viruses, para-influenza virus types
1-3, and adenovirus.
Clinical evaluation and statistical analysis.
The clinical diagnosis of ALRTI was based on the presence of
cough, tachypnea, chest indrawing, or wheeze of <7 days
duration, as per WHO standard protocol [12]. The medical files
of HCoV-NL63 infected children were written by pediatricians. In
brief, the lists included date of collection of NPA, demographic
data (age, gender, hospital days, history of wheezing of
patients or their families), symptoms (fever, cough, wheezing,
tachypnea, cyanosis, rhinitis, hoarseness of voice, vomiting and
diarrhea), laboratory testing (coinfection with viruses, white
blood cell count and differential count, C-reactive protein,
chest radiograph), discharge diagnosis, and outcome
(amelioration or recovery).
The study was approved by Children’s Hospital
of Chongqing Medical University.
Results
Sample collection. During the
study period, a total of 878 NPA from hospitalized children (592
males, 286 females) were collected, accounting for 13.9%
(878/6306) of the total admissions with ALRTI during the same.
The age of the patients ranged was ranging from 29 days to 15.9
years. As a result of unequal seasonal distribution of
patients with respiratory diseases, 44.4% of the total samples
were collected in winter (December to February); 19.1% from
March to May, 12.7% from June to August, and 23.8 % from
September to November, respectively.
Clinical features. All NPA were tested
for HCoV-NL63 by RT-PCR, and 8 out of 878 yielded positive
results, (Table I). Patients with HCoV-NL63 aged
from 1-8 mo, and the age of 6 positive patients was <6 months
(median, 4.6 months). RSV was detected positive by RT-PCR in 3
children with HCoV-NL63 infection and hMPV positive in 2
patients. None of the patients were detected positive for other
respiratory viruses. All HCoV-NL63 positive patients did not
have leucocytosis in peripheral blood tests, and none of
patients presented with leukopenia and lymphopenia. The
concentration of C-reactive protein was not elevated in 5 of 8
HCoV-NL63 patients tested for same. None of the 8 patients had
predisposing factors or underlying diseases, nor did they
require intensive care. Chest radiographs were done in 6 out of
8 children, all of them revealed varying degree of bilateral
infiltrates. Uneventful recovery or amelioration were achieved
in all patients.
TABLE I Clinical Features of Patients with HCoV-NL63 Infections
Patients |
Month
of NPA |
Hospital |
Age |
Sex |
Symptoms |
Coinfection |
Discharge diagnosis |
Outcome |
No. |
collection |
stay
(d) |
(mo) |
|
|
with
viruses |
|
|
1. |
September 2006 |
2 |
3.4 |
M |
Couth, fever, cyanosis, vomiting |
ND |
Bronchial pneumonia |
Amelioration |
2. |
September 2006 |
5 |
3.2 |
M |
Couth, rhinitis, trachyphonia |
ND |
Bronchitis |
Recovery |
3. |
November 2006 |
6 |
8.3 |
F |
Couth, fever, rhinitis |
RSV |
Bronchial pneumonia |
Recovery |
4. |
November 2006 |
10 |
4.0 |
M |
Couth, wheezing, fever, cyanosis |
RSC |
Bronchiolitis
tachypnea, rhinitis, diarrhea |
Recovery |
5.# |
*January 2007 |
4 |
5.1 |
M |
Cough, wheezing, tachypnea, cyanosis |
RSV, hMPV |
Interstitial pneumonia |
Amelioration |
6. |
August 2007 |
8 |
1.5 |
F |
Cough, tachypnea, cyanosis, vomiting, diarrhea |
ND |
Bronchial pneumonia |
Recovery |
7. |
*August 2007 |
8 |
7.9 |
M |
Couth, wheezing, tachypnea
rhinitis, vomiting, diarrhea |
hMPV |
Bronchiolitis |
Recovery |
8. |
September 2007 |
5 |
3.6 |
M |
Couth, cyanosis, tachypnea vomiting, diarrhea |
ND |
Bronchial pneumonia |
Amelioration |
* Family history of
wheezing present; # history of wheezing present in
child. |
Phylogenetic analysis. Nucleotide
sequences were analyzed for nucleotides 5778 to 6616 of the 1a
gene in these 8 positive specimens. Sequence comparison with
published Netherlands, Australia, Belgium, Canada, South Korea,
Sweden, South Africa and Italy isolates showed that the 1a genes
were relatively conserved, with nucleic acid identities ranging
from 95.3% to 100% among specimens. The phylogenetic tree of the
HCoV-NL63 isolates showed the existence of two major groups or
clusters, with the majority of isolates clustering in cluster 1.
In this study, four HCoV-NL63 isolates were found in cluster 1,
and the other four in cluster 2.
Discussion
In the study period, 8 out of 878 children
with ALRTI tested positive for HCoV-NL63, indicating a obviously
low prevalence of this virus as compared to other common
respiratory viruses such as RSV, hMPV, influenza A and B
viruses, para-influenza virus types 1-3, and adenovirus in
Southwest China; it was also lower than the reported prevalence
from other countries and regions (1.1% ~ 7%) [6,7,9,13-16].
Surprisingly, seasonality of HCoV-NL63 was
quite different from other respiratory viruses such as RSV,
which showed invariable peak in the winter months in Chongqing.
Some of the HCoV-NL63-positive cases; however, were identified
in hot and wet summer months in this region, indicating further
that long-term surveillance studies are needed to clarify
epidemiological picture of HCoV-NL63. In this study, all HCoV-
NL63 positive patients were under 12 months old, and most of
HCoV-NL63 positive patients (6/8) were under 6 months old. These
results show that HCoV-NL63 causes ALRTI in younger children,
especially in infants, as shown in previous studies [8,14].
In the present study, the clinical symptoms
associated with HCoV-NL63 were similar to those reported
previously and comparable to those observed in other viral
respiratory infections. Unlike previous reports that indicated
close association of HCoV-NL63 infection and croup, [17,18] none
of our patients presented with croup. In previous studies,
[8,9,11,15] some of the children with HCoV-NL63 infection
experienced underlying medical problems, especially prematurity
and pulmonary and cardiac diseases, whereas none of our patients
experienced underlying diseases identified. None of the 8
patients required intensive care, indicating that HCoV-NL63
infection does not lead to relatively severe illness requiring
internsive care even in infants. Unfortunately, the high
frequency of co-infections in HCoV-NL63 positive samples made it
difficult to define the clinical character of HCoV-NL63
infection.
The phylogenetic analysis of the HCoV-NL63
isolates indicated they belonged to the separated circulation of
cluster 1 viruses from September 2006 to December 2006, and
cluster 2 viruses from January 2007 to September 2007. A
one-year study from Canada also found all circulating isolates
were in cluster 1 [11], yielding a speculation that viruses from
one cluster may shift to another over time and therefore lead to
periodic circulation of two cluster viruses.
In conclusion, we confirmed the presence of
HCoV-NL63 in consecutive hospitalized children with ALRTI.
However, the association of this virus infection and respiratory
tract illnesses remains indistinct. Therefore, population-based,
prospective and multicenter study may be required to illustrate
the epidemiologic picture of HCoV-NL63 in China.
Acknowledgments: Professor Yan Li
(National Microbiology Laboratory, Canadian Science Center for
Human and Animal Health) for thoughtful and critical review of
the manuscript, and Mr. Qianglin Duan for critical reading of
the manuscript.
Contributors: CX: was responsible for
planning and conducting experiments, data analysis, and wrote
the manuscript; ZZY: took part in the collection of samples, RNA
extraction, cDNA synthesis and detection co-infection with RSV.
LY provided the sequence of primers used for HCoV-NL63 gene
amplification and gave some thoughtful and critical reviews of
the manuscript and ZXD: has designed research, provided project
guidance, suggested experimental design, reviewed data analysis
and interpretations, and critically revised the manuscript.
Funding: The National Natural
Science Foundation of China (No.30730098) and Natural Science
Foundation Project of CQ CSTC (No.cstc2011jjA10083).
Competing interests: None stated.
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