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Journal of Clinical Microbiology, July 2000, p. 2468-2474, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Determination of Human Immunodeficiency Virus Type
1 Subtypes in Taiwan by vpu Gene Analysis
Chun-Nan
Lee,1,2
Wei-Kung
Wang,3
Wen-Sheng
Fan,1
Shing-Jer
Twu,4
Shou-Chien
Chen,5
Ming-Ching
Sheng,2 and
Mao-Yuan
Chen6,*
School and Graduate Institute of Medical
Technology,1 Institute of
Microbiology,3 and Department of
Internal Medicine,6 College of Medicine, and
College of Public Health,4 National
Taiwan University, Department of Laboratory Medicine,
National Taiwan University Hospital,2 and
Taipei Municipal Venereal Disease Control
Institute,5 Taipei, Taiwan
Received 13 January 2000/Returned for modification 3 March
2000/Accepted 17 April 2000
 |
ABSTRACT |
The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of the env and gag genes. Information on the
vpu genes in the HIV sequence database is very limited. In
the present study, the nucleotide sequences of the vpu
genes were analyzed, and the genetic subtypes determined by analysis of
the vpu gene were compared with those previously determined
by analysis of the gag and env genes. The vpu genes were amplified by nested PCR of proviral DNA
extracted from 363 HIV-1-infected individuals and were sequenced
directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The vpu subtype of
one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of the env or gag gene. The genetic subtypes
determined by vpu gene analysis were, in general,
consistent with those determined by gag and/or env gene analysis except for those for two AG recombinant
strains. All the strains that clustered with a Thailand subtype E
strain in the vpu phylogenetic analyses were subtype E by
env gene analysis and subtype A by gag gene
analysis. In summary, our genetic typing revealed that subtype B
strains, which constituted 73.8% of all strains analyzed, were most
prevalent in Taiwan. While subtype E strains constituted about
one-quarter of the viruses, they were prevalent at a higher proportion
in the group infected by heterosexual transmission. Genetic analysis of
vpu may provide an alternate method for determination of
HIV-1 subtypes for most of the strains, excluding those in which
intersubtype recombination has occurred.
| |
INTRODUCTION |
Prominent genomic heterogeneity is
found among different human immunodeficiency virus (HIV) type 1 (HIV-1)
isolates. Phylogenetic analyses of env gene sequences from
different isolates obtained worldwide have identified at least 10 genetic subtypes (subtypes A to J) in the major (M) group and sets of
strains in the outlier (O) and new (N) groups (8, 14, 17, 19, 20,
24). The genetic diversity of HIV-1 is generated by accumulation
of point mutations and by recombination (9, 15).
In addition to gag, pol, and env, the
HIV-1 genome contains six accessory genes (18). One of them
is the vpu gene, which is located in the middle part of the
genome. vpu encodes a small phosphorylated protein, Vpu,
which is composed of the N-terminal transmembrane domain and the
C-terminal cytoplasmic domain. The cytoplasmic domain contains two
highly conserved serine residues that are phosphorylated
(25). Structure-function studies of Vpu revealed that the
transmembrane domain is critical for virus release and that the
cytoplasmic domain is required for degradation of CD4 (25).
The 3' one-third (83 bp for most of the HIV-1 reference strains) of the
vpu gene overlaps the env gene.
Information on the genetic subtypes of HIV-1 is important for
understanding of the global evolution of HIV-1 and for vaccine development. Genetic subtypes may also have an impact on drug susceptibility, as well as on the determination of drug resistance mutations and the measurement of viral loads (5, 7,
23). The genetic diversity of HIV-1 has mainly been characterized
by analysis of the env and gag genes (17,
22). Information on the sequence variation and the subtypes of
the vpu gene is still very limited. In the study described
here, the nucleotide sequences of the vpu genes were
analyzed. The genetic subtypes of HIV-1 determined by analysis of the
vpu gene were compared with those previously determined by
analysis of the gag and env genes (C. N. Lee, M. Y. Chen, C. L. Kao, H. S. Lin, M. C. Lee,
S. J. Twu, R. Y. Lin, M. C. Sheng, and C. Y. Chuang, Program Abstr. 4th Int. Conf. AIDS, abstr. V8, p. 22, 1996).
The prevalence of HIV-1 subtypes among different risk groups in Taiwan
was also determined on the basis of vpu sequence analysis.
| |
MATERIALS AND METHODS |
Samples.
HIV-1-positive blood samples have been collected
since 1990 at the Taipei Municipal Venereal Disease Center and National
Taiwan University Hospital. Blood samples were collected in sterile
EDTA-containing tubes. Peripheral blood mononuclear cells (PBMCs) were
separated by incorporating a Ficoll-Hypaque gradient (21).
For each HIV-1-infected individual, information including gender, age,
race, sexual orientation, risk factors, etc., was obtained through a
questionnaire, and the risk factors were rechecked by counseling with
medical personnel. The risk groups were categorized into hemophiliac,
intravenous drug user, and sexual transmission groups. The sexual
transmission group was further divided into female and male
heterosexual, homosexual, and bisexual groups. The differences in the
prevalence of HIV-1 subtypes between different groups were analyzed by
the 
2 test.
Genomic DNA isolation.
PBMCs were first treated with
erythrocyte lysis buffer (0.32 M sucrose, 10 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1% Triton X-100). The supernatant was discarded
after centrifugation. The pelleted cells were treated with proteinase K
(100 µg/ml) in a solution containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 2.5 mM MgCl2, 0.45% Nonidet P-40, and 0.45% Tween 20 at 55°C for 1 h. The DNA was then phenol-chloroform extracted,
ethanol precipitated, and quantitated by measuring the optical density
at 260 nm.
PCR.
The vpu genes were amplified by nested PCR.
One to 1.5 µg of genomic DNA extracted from PBMCs was used as the
template for the first round of PCR. The reaction mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% (wt/vol) gelatin, 1.5 mM
MgCl2, each of the deoxynucleoside triphosphates at a
concentration of 200 µM, 15 pmol of each of the primers, and 1 U of
Super Taq DNA polymerase (HT Biotechnology, Cambridge,
England). A close examination of the sequences available in the HIV
sequence database was carried out to identify highly conserved regions
that flank the vpu gene. Primer pairs that covered the
conserved regions were thus designed. They were TAT-1
(5'-CCTAAACTAGAGCCCTGGAACCATCC-3'; positions in SF2,
nucleotides 5855 to 5880) and EN70 (5'-GGTACACAGGCATGTGTGGCCC-3'; nucleotides 6435 to 6456) for the first-round PCR. The
amplification conditions were 30 cycles of 94°C for 30 s, 50°C
for 30 s, and 72°C for 30 s and a final extension at 72°C
for 10 min. The formulation for the second-round PCR was identical to
that for the first-round PCR, and the second-round PCR was performed in
a manner identical to that for the first-round PCR. A 1-µl aliquot of
the first-round PCR product was applied. The primers used in the
second-round PCR were 154.1 (5'-CTTAGGCATCTCCTATGGCAGGAAGAAG-3';
nucleotides 5965 to 5992) and KPN
(5'-ACACAGGTACCCCATAATAGACTGT-3'; nucleotides 6338 to 6362).
To avoid contamination and the resultant false-positive results,
standard precautions recommended for PCR were taken during the
amplification (11).
Purification of PCR product.
Five microliters of the final
PCR product for each sample was first checked in a 1% agarose gel.
Then, the amplified 398-bp fragments were purified through a QIAquick
silica gel membrane (QIAGEN, Chatsworth, Calif.).
DNA sequencing.
The purified PCR product was sequenced by
using the sequencing kit with fluorescent dye terminators
(Perkin-Elmer, Foster City, Calif.) according to the manufacturer's
instructions. The PCR primers were also used for sequencing. The
thermal cycling reactions were 96°C for 30 s, 50°C for 15 s, and 60°C for 4 min for 25 cycles. The products were purified by
ethanol precipitation and were then resuspended in the loading buffer
(formamide and 25 mM EDTA [5:1]). The samples were heated at 90°C
for 2 min and were loaded onto a 4.75% polyacrylamide gel. The
sequence data were collected with an autosequencer (ABI-373A;
Perkin-Elmer).
Analysis of sequences.
The full-length vpu genes
were aligned with the vpu genes of selected reference
strains that represented various HIV-1 subtypes from the Los Alamos HIV
database. The sequence data were analyzed with GeneWorks software
(IntelliGenetics, Mountain View, Calif.). The phylogenetic
relationships of the vpu genes among HIV-1 strains were
analyzed by the neighbor-joining method and the Kimura 2-parameter distance matrix listed in the MEGA (molecular evolutionary genetic analysis) analytical package (10). The sequences of
following reference strains (with their subtypes, GenBank accession
numbers given in parentheses) were used for comparison: U455 (A,
M62320), UG273 (A, L22957), UG275 (A, L22951), 92UG037 (A, U51190), SF2
(B, K02007), MN (B, M17449), JRFL (B, U63632), ETH2220 (C, U46016),
92BR025 (C, U52953), NDK (D, M27323), Z2Z6 (D, M22639), CM240X (AE,
U54771), BZ126A (F, L22082), BZ163A (F, L22085), HH8793 (G, AF061641),
SE6165 (G, AF061642), 92NG083 (AG, U88826), IBNG (AG, L39106), 90CR056
(H, AF005496), ANT70 (group O, L20587), and MVP5180 (group O, L20571).
Nucleotide sequence accession numbers.
The vpu
gene sequences determined in this study were deposited in the GenBank
sequence database. The accession numbers are AF143901 to AF143903 for
TWA strains, AF220462 to AF220471 for TWB strains, AF220472 to AF220475
for TWC strains, AF220476 to AF220485 for TWE strains, and AF220486 to
AF220489 for TWG strains.
| |
RESULTS |
Determination of vpu subtypes.
The vpu
genes of HIV-1 strains from 363 infected individuals in Taiwan were
amplified by PCR and completely sequenced. The nucleotide sequences
were aligned with reference strains of subtypes A, B, C, D, E, F, G,
and H and were subjected to phylogenetic analysis. The genetic subtypes
of the vpu genes were thus determined on the basis of the
alignments and the phylogenetic trees. A typical phylogenetic tree with
some of the strains is shown in Fig. 1. These Taiwanese strains were designated "TW," followed by
characters that indicate the env subtype. An env
subtype A variant, TWA1, clustered with subtype A reference strains
U455, UG273, UG275, and 92UG037 (Fig. 1). Strains TWB64, TWB93, TWB101,
TWB128, TWB129, TWB136, TWB171, TWB207, TWB219, and TWB220 clustered
with subtype B reference strains SF2, MN, and JRFL. Strains TWC1 to
TWC4 clustered with subtype C reference strains 92BR025 and ETH2220.
Strains TWE2, TWE5, TWE6, TWE13, TWE14, TWE23, TWE30, TWE31, TWE57, and TWE58 clustered with subtype E reference strain CM240X. Strains TWG1 to
TWG4 and TWA3 formed a cluster with subtype G reference strains HH8793
and SE6165 and with AG recombinants IBNG and 92NG083. It should be
noted that the subtype E strains form a distinct group, although this
group is also closely related to subtype A strains (Fig. 1). The
vpu subtype of one strain, TWA2, could not be determined,
since it did not cluster with any of the strains analyzed.
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FIG. 1.
Scaled vpu gene neighbor-joining phylogeny of
31 Taiwanese strains and 19 reference sequences from various HIV-1
subtypes, with ANT70 and MVP5180 as the outliers. The Taiwanese strains
are indicated by boldface italic type. The Taiwanese strains were
designated according to the env subtype, which is indicated
by the characters that follow "TW." Bootstrap values above 60% are
indicated at the branch nodes. A branch length nucleotide difference of
10% is indicated at the bottom.
|
|
Sequence analysis of vpu genes.
Sequence analysis
of the vpu genes of three previously reported Taiwanese
env subtype A variants (12), TWA1, TWA2, and
TWA3, revealed that the sequence divergences among them were greater than 20%. The results of the sequence comparisons of the
vpu genes of these env subtype A variants and
some of the Taiwanese strains of different subtypes are summarized in
Table 1. The nucleotide sequence
divergences among strains of each of the env subtypes B, C,
E, and G were less than 14%, whereas the divergences among strains of
different env subtypes were generally greater than 17%. The
vpu gene of TWA3 was classified as subtype G in the
phylogenetic analysis, but it varied from those of the other four
Taiwanese subtype G strains that were epidemiologically linked
(13) (divergence range, 13.4 to 14.6%). TWA1 did not show a
high degree of similarity to the other Taiwanese strains. However, its
vpu gene was closely related to those of subtype A reference
strains U455, UG273, UG275, and 92UG037, with similarities ranging from
85.8 to 90.7%. This was consistent with the classification of TWA1 as
vpu subtype A in the phylogenetic analysis. As for the
strain with an undetermined vpu subtype, TWA2, its
nucleotide sequence did not show a high degree of similarity to that of
any Taiwanese strain or to those of any of the subtype reference
strains analyzed (Table 1 and Table
2).
The deduced amino acid sequences of the
vpu gene were also
aligned and analyzed. As shown in Fig.
2,
two serine residues were
retained by all of the strains analyzed. In
addition, charged
residues were highly conserved at 11 positions. The
strains of
the same subtype were more similar in terms of the deduced
amino
acid sequences of Vpu. Of note was the fact that the sequence
of
the C-terminal one-third of TWA2 was similar to those of CM240X,
HH8793, and SE6165. In particular, of the 27 amino acid residues
in
this region (from residue N55 to residue L81), TWA2 was identical
to
CM240X at 26 positions. Since the C-terminal one-third but
not the
N-terminal portion of the Vpu amino acid sequence of TWA2
was very
similar to those of some of the reference strains, the
nucleotide
sequence similarity between TWA2 and the subtype reference
strains was
reexamined by dividing the sequence into two portions:
the 5'
two-thirds and 3' one-third. As shown in Table
2, the
3' one-third of
TWA2 had 91.6% or greater similarity to the 3'
one-third of HH8793,
SE6165, 92NG083, and CM240X, whereas the
similarity of the 5'
two-thirds of TWA2 to the 5' two-thirds of
any of the strains analyzed
was less than 80% (Table
2).
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FIG. 2.
Deduced amino acid alignment of Vpu for the 31 Taiwanese
strains and 9 reference strains. The vpu subtypes of the
reference strains are indicated in parentheses. The alignment was
constructed with GeneWorks software (IntelliGenetics, San Jose,
Calif.). The consensus sequence inferred from the 40 sequences is shown
at the top of the alignment. Dots indicate identity with the consensus
sequence. Dashes denote gaps generated during the alignment. Conserved
serine residues are indicated by asterisks.
|
|
Comparison of HIV-1 subtypes determined by different genes.
The genetic subtypes determined by vpu gene analysis were
compared with those previously determined by env and
gag gene analysis (Lee et al., Program Abstr. 4th Int. Conf.
AIDS). Table 3 summarizes the results of
the comparison for the strains in 247 samples. Of the strains in these
samples, both the env and gag subtypes were
determined for 69 strains, only the env subtype was
determined for 143 strains, and only the gag subtype was
determined for 35 strains. As shown in Table 3, the genetic subtypes
determined by vpu gene analysis were generally in agreement
with those determined by env and/or gag gene
analysis. Of the 212 strains for which both vpu and
env subtypes were available, 210 were of the same subtype.
Of the 104 strains for which both vpu and gag
subtypes were available, 65 were of the same subtype but 37 were of
subtype E by vpu analysis and subtype A by gag
analysis. The 37 subtype E vpu strains were actually AE
recombinants which contain subtype E env and subtype A
gag.
vpu subtypes of the HIV-1 strains among different risk
groups in Taiwan.
Of the 363 samples collected from HIV-1-infected
individuals, the strains in 268 (73.8%) were classified as subtype B,
the strains in 84 (23.1%) were classified as subtype E, the strains in
5 (1.4%) were classified as subtype G, the strains in 4 (1.1%) were
classified as subtype C, the strain in 1 (0.3%) was classified as
subtype A, and the strain in 1 was unclassified (0.3%). The prevalence
of HIV-1 subtypes among different risk groups in Taiwan was further
analyzed and is summarized in Table 4.
Eleven HIV-1-seropositive hemophiliacs who had received coagulation
factors were all infected with subtype B viruses. Of the 10 intravenous
drug users, 7 were infected with subtype B viruses and 3 were infected
with subtype E viruses. Of the 35 strains collected from women who were
not intravenous drug users and who were infected through heterosexual contact, 10 (28.6%) were subtype B, 21 (60.0%) were subtype E, 3 (8.6%) were subtype G, and 1 (2.9%) was unclassified. The group of
men who were not intravenous drug users but who were infected with
HIV-1 by sexual transmission included 307 individuals. Two hundred
forty (78.2%) were infected with subtype B viruses and 60 (19.5%)
were infected with subtype E viruses. Among the individuals in this
risk group, all 78 homosexual men were infected with subtype B viruses.
Of the 136 heterosexual men, 82 (60.3%) were found to be infected with
subtype B virus, 47 (34.6%) were infected with subtype E virus, and
the rest of them were infected with either subtype A, subtype C, or
subtype G virus. These results indicated that while subtype B infection
was predominant in the whole study population as well as in most of the
risk groups, subtype E viruses constituted a significant proportion of
the viruses in the heterosexual transmission risk group. Compared with
the prevalence of subtype E infection in homosexual and bisexual men,
the prevalence of subtype E infection in heterosexual men was much
higher (P < 0.005 by the 2 test).
Furthermore, subtype E infection was prevalent at a significantly higher proportion in the women, who were infected primarily through heterosexual contacts, than in the men (P < 0.0001 by
the 2 test).
| |
DISCUSSION |
We have used vpu gene sequencing analyses to subtype
363 HIV-1-positive samples. Only one strain could not be subtyped in this manner. The possibility of coinfection with two subtypes of HIV-1
in one individual was remote, since direct sequencing of each PCR
product as described in this study revealed no evidence that indicated
the coexistence of two distinct sequences. Furthermore, the
vpu subtypes were generally in agreement with those
determined by analysis of the env or gag gene.
There was one exception, in which the subtype determined by
vpu gene analysis was different from the subtype determined
by env and gag gene analyses. This strain (TWA3),
which was subtype A by gag and env gene analyses, as identified previously, was found in this study to be of subtype G by
vpu gene analysis (12). Therefore, TWA3 was an AG recombinant.
The one strain (TWA2) that had an undetermined vpu subtype
in this study was previously characterized as an intersubtype (AG) recombinant with subtype G by gag gene analysis and subtype
A by env gene analysis (12). TWA2 did not show a
high degree of similarity to any subtype reference strains analyzed.
However, the 3' one-third of the vpu gene of TWA2 had a
higher degree of similarity to those of subtype G and subtype E strains
than to those of strains of other subtypes. These observations suggest that part of the vpu gene of TWA2 can be classified as
subtype G. Of note is that CM240X, an AE recombinant from Thailand, is closely related to subtype G strains in the 3' one-third of
vpu, as revealed by nucleotide sequence alignment (with
91.6% similarity) and by phylogenetic analysis (data not shown).
CM240X was known to have a subtype A gag gene and a subtype
E env gene (1). The 3' one-third of
vpu is the region of vpu that overlaps
env. Although the reason for the clustering of subtype E and
subtype G in this region remains unclear, the close relationship
between subtype E and subtype G has also been described for the
cytoplasmic domain of gp41, and subtype E and subtype G strains form a
single cluster in the coding region for the cytoplasmic domain of gp41 by phylogenetic analysis. (2).
The vpu genes of the Taiwanese env subtype E
strains were closely associated with that of CM240X by phylogenetic
analysis. These subtype E strains form a distinct group which was close to the subtype A strains in the phylogenetic tree. While the
similarities of the vpu genes of these subtype E strains to
those of subtype A strains U455, UG273, UG275, and 92UG037 ranged from
81.7 to 85.0%, the similarity of the vpu genes of these
strains to that of CM240X was in the range of 93.9 to 98.0%.
Therefore, the vpu genes of the subtype E strains could
easily be distinguished from those of the subtype A strains.
The epidemic of HIV-1 infection in Taiwan began at about the same time
that HIV-1 was introduced into Asia (16, 26). The first case
of HIV-1 infection in Taiwan was reported in 1985. Since 1991, the
seroprevalence rates have increased rapidly. As the end of November
1999, 2,375 cumulative HIV-1 infections had been reported to the
Department of Health (Center for Disease Control, Department of Health,
Taiwan, Republic of China, http://www.cdc.gov.tw/e/aids). Among
them, 982 were among individuals in the risk category of heterosexual transmission, 664 were among homosexual men, and 383 were
among bisexual men (Center for Disease Control, Department of Health).
Although the main risk factor has been reported to shift from
homosexual to heterosexual contact in 1992 (3), an
extraordinary male-to-female ratio of 12:1 was noted.
The spread of subtype B HIV-1 in Taiwan was first found in the
homosexual male population through contact with foreigners. Later,
subtype E virus was introduced into Taiwan through travelers who had
heterosexual contact with commercial sex workers in Thailand. This
study revealed that subtype B was the most prevalent subtype in Taiwan,
whereas subtype E viruses constituted a significant proportion of the
HIV-1 strains that infected those in the heterosexual transmission risk
group. These results were generally in agreement with the findings of a
previous study which determined the subtypes by the peptide serotyping
method (4). However, that study showed a much higher
proportion of subtype E virus in the homosexual male population
(20.8%). This discrepancy could partially be explained by the
cross-reactivity by the peptide serotyping method and by the limited
number of samples selected. To clarify this, more samples need to be
studied in the future.
The genetic variability of HIV-1 has been a problem for PCR
amplification. It is difficult to design a single primer set in the
env and gag regions suitable for detection of all
HIV-1 strains. Multiple primer pairs and different amplification
conditions are commonly used to achieve the optimal results
(6). However, PCR amplification of the vpu gene
with the primers and the conditions reported in this study was rather
satisfactory. This was probably due to the conservation of the
sequences that flank the vpu gene. In addition, the
specificities of the primers were validated by the findings that each
positive PCR product was completely sequenced and was found to be
a vpu sequence. Furthermore, the vpu gene of the
virus in each sample occupied a distinct position in the phylogenetic
tree, suggesting that cross-contamination between samples was unlikely
to have occurred. The sensitivity of the PCR protocol described here
was good, since the detection limit was estimated to be four copies of
HIV-1 DNA spiked in genomic DNA extracted from seronegative PBMCs (data
not shown). PCR amplification of vpu was promising at least
for our local circulating strains of subtypes A, B, C, E, and G. vpu gene analysis may provide additional information and an
alternate method from traditional gag and env analysis for determination of the subtypes of most of the HIV-1 strains, excluding those in which intersubtype recombination has occurred.
| |
ACKNOWLEDGMENTS |
We are grateful to the staff of Taipei Municipal Venereal Disease
Control Institute for the collection of samples, Hui-Shiuh Lin and
Shih-Hsiang Chien for technical assistance, and Chin-Der Lee for data analysis.
This work was supported by grants from the National Taiwan University
Hospital (grants NTUH-85209-B01 and NTUH-87S1522) and the Department of
Health (grant DOH87-TD-1038) of Taiwan, Republic of China.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Internal Medicine, College of Medicine, National Taiwan University, No. 7, Chung-Shan South Rd., Taipei, Taiwan 100, Republic of China. Phone:
886-2-23123456, ext. 5030. Fax: 886-2-23223905. E-mail: moyc{at}ha.mc.ntu.edu.tw.
| |
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Journal of Clinical Microbiology, July 2000, p. 2468-2474, Vol. 38, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
