Molecular Epidemiology Of Hiv 1 Variants Biology Essay

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In this study, 1045 HIV-1 positive patients who have not received any antiretroviral therapy were recruited from January 2003 to December 2008. This patient collection represented 51.4% (1045/2032) of all new diagnoses of HIV-1 infection in Hong Kong until the end of 2008.

The pol sequences were obtained from routine GRT of individual patients to investigate the distribution of HIV-1 Group M variants in Hong Kong. From the pol phylogenetic analyses, 96.7% of samples (1011/1045) could be assigned with a confident genotype in which subtype B (450/1045; 43.1%) and CRF01_AE (469/1045; 44.9%) were the predominant genotypes among the sample collection. HIV-1 variants with other genotypes were recognized in 8.8% of samples (92/1045) including subtype A1 (1/126; 0.8%), C (44/126; 34.9%), D (1/126; 0.8%), CRF02_AG (7/126; 5.6%), CRF03_AB (2/126; 1.6%), CRF06_cpx (1/126; 0.8%), CRF07_BC (20/126; 15.9%), CRF08_BC (9/126; 7.1%), CRF15_01B (2/126; 1.6%), CRF22_01A1 (1/126; 0.8%), CRF33_01B (2/126; 0.8%), CRF42_02G (1/126; 0.8%) and CRF43_02G (1/126; 0.8%)(Table 3.1).

Apart from the samples with confident HIV-1 genotype, there were 34 samples (34/1045; 3.3%) that could not be genotyped in pol phylogenetic analyses. The NJ phylogenetic tree constructed using pol sequences of these 34 samples was presented in Figure 3.1. It is revealed that the samples were randomly disturbed throughout the NJ tree and could not be clustered to any known subtypes or CRFs reference sequences with high bootstrap value (>70) or be formed an out-group from subtypes or CRFs reference sequences.

Table 3.1. The distribution of HIV-1 Group M variants in Hong Kong.

HIV-1 genotype

n

Prevalence %

CRF01_AE

469

44.9%

Subtype B

450

43.1%

Other variants

92

8.8%

A1

1

0.8%

C

44

34.9

D

1

0.8%

CRF02_AG

7

5.6%

CRF03_AB

2

1.6%

CRF06_cpx

1

0.8%

CRF07_BC

20

15.9%

CRF08_BC

9

7.1%

CRF15_01B

2

1.6%

CRF22_01A1

1

0.8%

CRF33_01B

2

0.8%

CRF42_02G

1

0.8%

CRF43_02G

1

0.8%

Sample with unassigned genotype

34

3.3%

Total

1045

100%

Figure 3.1. The neighbor joining phylogenetic tree of pol sequence from 34 samples with undefined HIV-1 genotypes (labeled in Blue). Bootstrap values (1000 replicates) larger than 50 were shown next to the nodes of the tree. Bar, 0.02 substitutions per site.

env Genotyping of HIV-1 Variants with undefined genotype

env Phylogenetic analyses

For those 34 samples presenting undefined HIV-1 genotype in routine pol phylogenetic analyses, classical env genotyping method targeting C2V3V4 region of gp41 protein was performed. The results of env phylogenetic analyses were used for the sample selection of genomic analyses.

Throughout the env phylogenetic analyses, 26 out of 34 samples (76.5%) were successfully amplified and sequenced. The remaining 8 samples were repeated failure in either PCR amplification or DNA sequencing of env gene. From the NJ phylogenetic tree in Figure 3.2, a confident HIV-1 genotype could be assigned to 14 samples (14/34; 41.2%). Among these 14 samples, different subtypes and CRFs could be found which contained subtype A (2/34; 5.88%), subtype B (4/34; 11.76%), CRF01_AE (6/34; 17.65%), CRF02_AG (1/34; 2.94%) and CRF08_BC (1/34; 2.94%). Besides, the result of undefined HIV-1 genotype was still found in 12 samples (35.29%) which could not be clustered to any known subtypes or CRFs reference sequences with high bootstrap value (>70) or be formed an out-group from subtypes or CRFs reference sequences. These 12 samples were selected as the candidates of HIV-1 genomic analyses.

Identification of Interesting Clusters and Samples for HIV-1 Genomic Analyses

Due to the limited resources and high reagent cost, full-length sequencing of HIV-1 genome could not be performed on all 12 samples presenting undefined HIV-1 genotype in both pol and env phylogenetic analyses. With this reason, only those samples presenting interesting results in env phylogenetic analyses or having special epidemiological background were selected for further analyses.

From the NJ tree of env phylogenetic analyses, a cluster of 5 isolates which formed as an out-group from the reference sequences of subtype B were identified (Figure 3.3). No reference sequence was found in this cluster. The presence of this cluster in the NJ tree was supported by a bootstrap value of 100. Since the presence of this cluster indicated the possibility of identification of URFs or even new CRFs, these 5 isolates were selected for the HIV-1 genomic analyses.

Besides this cluster, another sample was included in the full-length sequence analyses of HIV-1 genome because of the special epidemiological background presented by this sample. This patient is a non-Chinese Asian female who infected with HIV by the route of heterosexual. The plasma sample of this patient was collected in 2004. Since the circulation of URFs in out region may be introduced by the immigrant from other Asian countries, it would be interesting to include this sample in our study.

As the result, 6 samples were selected for the HIV-1 genomic analyses. In order to facilitate the naming in further analyses, sample number was assigned to each sample. Sample number 451, 737, 1009, 1325 and 1386 were used to represent the 5 samples in the cluster of NJ tree while sample number 512 was used to represent the sample with special epidemiological background.

Figure 3.2. The neighbor joining phylogenetic tree of env sequence from 34 samples with undefined HIV-1 genotypes (labeled in Blue). Bar, 0.03 substitutions per site.

Cluster selected for genomic analyses

Figure 3.3. The close-up view of the env neighbor joining phylogenetic tree showing the cluster of 5 sequences as an out-group from subtype B reference sequences (highlight in red rectangle). The cluster was supported with bootstrap value of 100. Bar, 0.03 substitutions per site.

Epidemiological Analyses and Genomic Characterization of HIV-1 URFs in Hong Kong

Optimization of HIV-1 Full-length Genome Sequencing System

Before starting the analyses of epidemiological relationship and genomic characterization of HIV-1 URFs in our region, the optimization of the in-house genomic sequencing system was necessary for the success of analyses. As the quality of cDNA from the viral RNA genome played an important role in the amplification and downstream sequencing of viral genome, the optimization was focused on the reaction of reverse transcription. In this study, two different components in the reverse transcription of the in-house genomic sequencing system were optimized to increase the sensitivity and specificity of the amplification of HIV-1 URFs.

For the primer used in reverse transcription, three different types of oligonucleotide that recommended by the manufacturer were subject for the optimization. The results revealed that the performance of Oligo dT and gene specific primer were better than random hexamers in which negative result was commonly found in all three PCR reactions of the in-house full genome amplification using random hexamers for the reverse transcription. Besides, the use of gene specific primer was found unsuitable in some clinical samples, especially the samples with HIV-1 genotype other than subtype B.

For the amount of reverse transcriptase employed in the reaction, 200 U of reverse transcriptase was originally used in the reaction as recommended by the manufacturer. However, the increased input of reverse transcriptase from 200 U to 400 U was significantly improved the efficacy of RT-PCR, especially in the samples with HIV-1 viral load of 500 and 1000 copies/mL.

From the result of optimization, it found that the use of Oligo dT as the primer and the employment of double amount of reverse transcriptase were much appropriated in the in-house full-length genome sequencing system.

Performance of in-house Full-length Genome Sequencing System

The performance of in-house full-length genome sequencing system on the 6 selected isolated was evaluated. From the result of PCR-sequencing, 5 out of 6 samples were successfully amplified and sequenced by the in-house system. For the sample (sample 1386) showing failure result, it revealed that multiple bands were found in gel electrophoresis result of F3 PCR reaction. Although sequencing reactions were still carried out for this PCR reaction, failure sequencing results with mixed nucleotide bases were found. In this case, sample 1386 was excluded from this study as the viral genomic sequence cannot be retrieved by the in-house system.

Epidemiological Analyses

Before starting the phylogenetic analyses and characterizing the genomic structure of the 5 isolates that suspected to be HIV-1 URFs in our region, their epidemiological backgrounds were collected and analyzed (Table 3.1). Sample 451 was a Chinese male patient who infected with HIV-1 by MSM transmission and diagnosed as HIV-1 carrier in 2004. It was reported that he had bisexual behavior with sexual contacts in Hong Kong. Sample 512 was the only non-Chinese Asian patient included in the study. She was the heterosexual female who was diagnosed HIV-1 positive in 2003 and the plasma sample was collected in 2004. Sample 737 was Chinese male and confirmed catching disease through heterosexual transmission. The epidemiological background showed that he was confirmed as HIV-1 carrier in 2005 and the amount of virions in plasma was in low level. Both sample 1009 and 1325 were Chinese male who infected with HIV-1 by the route of MSM. The first one was diagnosed HIV-1 positive in 2006 while the latter one was found to be HIV-1 carrier in 2008.

Table 3.1. Epidemiological backgrounds of 6 HIV-1 patients that their viral isolates subject to genomic analyses in this study

Sample No.

Ethnicity / Gender

Age

Route of Infection

1st serology positive date

Plasma sampling date

Viral load (copies/mL)

CD4+ (cells/mm3)

451

Chinese / Male

36

MSM

March 2004

March 2004

22000

407

512

Non-Chinese / Female

39

Heterosexual

July 2003

July 2004

320000

353

737

Chinese / Male

39

Heterosexual

October 2005

October 2005

<400

127

1009

Chinese / Male

41

MSM

December 2006

December 2006

30000

234

1325

Chinese / Male

36

MSM

January 2008

February 2008

880000

256

Phylogenetic Analyses of HIV-1 URFs in Hong Kong

By plotting the NJ phylogenetic tree with two reference sequence sets, 5 isolates were subject to phylogenetic analyses to study the prevalence of HIV-1 URFs in Hong Kong.

From the NJ phylogenetic tree using Los Alamos reference sequence set (Figure 3.4), a cluster of sequences which including sample 737, 1009 and 1325 was identified. No reference sequence was found in the cluster. This cluster formed as an out-group from subtype B reference sequence which supported by the bootstrap value of 100. Secondary transmission was observed within these 3 isolates in which sample 737 and 1009 were grouped together to form a sub-cluster while sample 1325 was found to be branch out from this sub-cluster. Apart from this finding, sample 451 was genotyped as CRF15_01B which supported by the bootstrap value of 100 while sample 512 was genotyped as CRF01_AE-like outgroup.

Another NJ phylogenetic tree using NCBI 2009 reference sequence set was plotted (Figure 3.5). The presence of the cluster including 3 sequences (sample 737, 1009 and 1325) was still observed in this NJ phylogenetic tree. Just like the one of Los Alamos, the cluster in NCBI NJ tree also presented as the out-group of subtype B reference sequence with bootstrap value of 94 with similar sequence structure was found (sub-cluster of sample 737 and 1009 with a branch of sample 1325). It was also revealed that no reference sequence was found in this cluster. On the other hand, sample 512 was genotyped as CRF22_01A1 which supported by the bootstrap value of 100 while sample 451 was presented as the CRF15_01B-like outgroup.

Subtype B

Reference sequences

Figure 3.4. The neighbor joining phylogenetic tree of near full-length sequence from 5 isolates selected for genomic analyses (labeled in Blue) using reference sequence set of Los Alamos HIV Database. Bar, 0.02 substitutions per site.

Subtype B

Reference sequences

Figure 3.5. The neighbor joining phylogenetic tree of near full-length sequence from 5 isolates selected for genomic analyses (labeled in Blue) using reference sequence set of NCBI Viral Genotyping Tool. Bar, 0.03 substitutions per site.

Comparison of the NJ tree result of two Reference Sequence Sets

From the result of two NJ phylogenetic trees, the performance of two different reference sequence sets on the phylogenetic analyses of full-length HIV-1 genomic sequence was compared. For sample 737, 1009 and 1325, both NJ phylogenetic trees showed that these 3 isolates formed a cluster and presented of as subtype B-line out-group in which the presence of cluster on both NJ trees was supported by high bootstrap value (Los Alamos NJ tree: 100; NCBI NJ tree: 94). For sample 451, the result of Los Alamos NJ tree showed that this sample was genotyped as CRF15_01B while it was genotyped as CRF15_01B-like out-group in NCBI NJ tree. For sample 512, it was presented as CRF01_AE-like out group in Los Alamos NJ tree while it was genotyped as CRF22_01A1 in NCBI NJ tree. Since no reference sequence of CRF22_01A1 in the Los Alamos reference sequence set, it was believed that sample 512 should be genotyped as CRF22_01A1 rather than CRF01_AE-like outgroup.

Genomic Characterization of HIV-1 URFs in Hong Kong

Throughout the phylogenetic analyses of near full-length viral genome, sample 512 was believed to be HIV-1 strain of subtype CRF22_01A1 in the NJ tree using NCBI reference sequence set. Meanwhile, there were 4 samples still could not be assigned with confident genotype, even using two different reference sequence sets. As the result, genomic characterization of these 4 samples were studied, using bootscanning analyses with Simplot software (Salminen et al., 1995), to reveal if these samples were HIV-1 URFs and the degree of recombination in these samples.

The result of bootscaning analyses revealed that sample 451 and 1325 were found to have genetic recombination in the viral genome which involved fragments of subtype B, CRF01_AE and fragments that remain unclassified (U), while sample 737 and 1009 were found to be the new sub-subtype B which including alternating fragments of subtype B and U.

The recombination analyses of sample 451 showed that it was the recombinant of CRF01 and subtype B, UCRF01/B/U recombinant (Figure 3.6). This recombinant virus contained a backbone structure of CRF01_AE which spanning the gag, pol, vif, vpr, vpu and nef. The fragments of subtype B were found in fusion region of gag and pol, the region coding the protease in pol gene and coding gp120 surface protein in env gene. It was also noticed that there were 3 fragments of U found in the viral genome which located in regions of gag, pol and env.

For sample 737, the analyses revealed that this virus could be classified as the UB/U recombinant (Figure 3.7). The fragments of subtype B made up the backbone of viral genome while fragments of U were found to be located in different regions of viral genome including region coding structural matrix and capsid proteins in gag, reverse transcriptase and integrase in pol and gp120 surface protein and gp41 transmembrane protein in env.

Throughout the bootscaning analyses, sample 1009 was also classified as the UB/U recombinant (Figure 3.8). The recombination pattern of sample 1009 was found to be similar to that of sample 737 where the recombination breakpoints in the viral genome of sample 1009 were nearly same as sample 737, except the region coding integrase in pol and gp41 transmembrane protein in env.

The recombination pattern of sample 1325 was also found to be similar to sample 737 and 1009 except CRF01_AE was involved in the recombination (Figure 3.9). Apart from the fragments of subtype B and U that found in gag, pol, vif, vpr, vpu, tat and rev, the fragments of CRF01_AE were located in the region coding reverse transcriptase and integrase and region of nef. Therefore, this virus was classified as the recombinant of subtype B, CRF01AE and U.

B

A

Figure 3.6. Recombination analyses and genomic characterization of sample 451. (A) Bootscan plot of sample 451 in comparison to reference sequences of all HIV-1 group M pure subtypes and CRF01_AE(CRF01_AE in red, subtype B in green), using a window of 400 nucleotide segments and increments of 50 nucleotide. (B) Genomic characterization of sample 451 showing the recombination pattern based on CRF01_AE (in red), subtype B (in blue) and unclassified segments (U; in grey). The illustration was created according to HXB2 numbering using the Recombinant Drawing tool available on the Los Alamos HIV sequence Database website.

B

A

A

Figure 3.7. Recombination analyses and genomic characterization of sample 737. (A) Bootscan plot of sample 737 in comparison to reference sequences of all HIV-1 group M pure subtypes and CRF01_AE (subtype B in green), using a window of 400 nucleotide segments and increments of 50 nucleotide. (B) Genomic characterization of sample 737 showing the recombination pattern based on subtype B (in blue) and unclassified segments (U; in grey). The illustration was created according to HXB2 numbering using the Recombinant Drawing tool available on the Los Alamos HIV sequence Database website.

B

A

Figure 3.8. Recombination analyses and genomic characterization of sample 1009. (A) Bootscan plot of sample 1009 in comparison to reference sequences of all HIV-1 group M pure subtypes and CRF01_AE (subtype B in green), using a window of 400 nucleotide segments and increments of 50 nucleotide. (B) Genomic characterization of sample 1009 showing the recombination pattern based on subtype B (in blue) and unclassified segments (U; in grey). The illustration was created according to HXB2 numbering using the Recombinant Drawing tool available on the Los Alamos HIV sequence Database website.

A

B

Figure 3.9. Recombination analyses and genomic characterization of sample 1325. (A) Bootscan plot of sample 1325 in comparison to reference sequences of all HIV-1 group M pure subtypes and CRF01_AE (subtype B in green), using a window of 400 nucleotide segments and increments of 50 nucleotide. (B) Genomic characterization of sample 1325 showing the recombination pattern based on CRF01_AE (in red), subtype B (in blue) and unclassified segments (U; in grey). The illustration was created according to HXB2 numbering using the Recombinant Drawing tool available on the Los Alamos HIV sequence Database website.

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