The Distribution of Human Papillomavirus Type 30 (HPV30) Lineages Among Women With Normal Cervical Cytology in China


 Background: Human papillomavirus type 30 (HPV30) is involved in cervical diseases. In human immunodeficiency virus (HIV) positive women, the prevalence HPV30 is almost the same as HPV16. However, HPV30 has seldom been investigated. In order to better understand the prevalence and intratype lineage distribution of HPV30 in China, HPV30 infection among women with normal cytology was investigated.Methods: Prevalence of HPV30 was investigated by the screening of type specific polymerase chain reaction (PCR); intratype lineage distribution was performed by the phylogenetic analysis of the L2 DNA sequences of HPV30 isolates; the diversity of genetic variants of HPV30 isolates was also evaluated. Results: (1) The infection rate of HPV30 was 0.56% (9/1600). (2) All the nine HPV30 isolates belonged to lineage A, none belonged to lineage B. (3) Compared with the HPV30 prototype reference, 87 variations including 79 substitutions, four insertions and four deletions were observed in this study. (4) Sample 4 contained a C base deletion of the E2 gene resulting in an amino acid sequence shift. (5) Sample had a truncated L2 protein.Conclusions: The infection rate of HPV30 is 0.56% in this study. All of the HPV30 isolates belongs to lineage A. Natural L2 and E2 defective isolates of HPV30 were found.


Background
Cervical cancer (CC) is one of the most common malignant cancers among women worldwide 3,4 . If the primary and secondary prevention programs are not carried out in low-income and middle-income countries, over 44 million women will be diagnosed as CC in the next 50 years 5 . The oncogenic human papillomavirus (HPV) types associated with all CCs are phylogenetically clustered in one clade includes of species Alpha-5 (A5), Alpha-6 (A6), Alpha-7 (A7), Alpha-9 (A9) and Alpha-11 (A11) of the Alpha genera 6 . Among the A6 HPVs, relatively high prevalence of Human papillomavirus type 53 (HPV53), Human papillomavirus type 56 (HPV56) and Human papillomavirus type 66 (HPV66) were investigated in the research on worldwide distribution of HPV in 2005 which account for about 6%, 36% and 21% infection rate that of Human papillomavirus type 16 (HPV16), respectively. Human papillomavirus type 30 (HPV30) was originally detected in a tissue specimen from laryngeal carcinomas, and was also detected in genital lesions 7 . In taxonomy, HPV30 is classi ed into the A6 specie of Alpha genera together with HPV53, HPV56 and HPV66 as well. But HPV30 is always a relatively rarely reported genotype worldwide, with few data available to characterize its prevalence, lineage distribution, and disease association.
In recent years, with the double detection of Human immunode ciency virus (HIV) and HPV, it has been found that the prevalence of HPV in HIV positive women is higher than in HIV negative women. Especially in HIV positive women, the prevalence of HPV30 is almost the same as HPV16 1,2 , which suggests the importance of the research of HPV30. This may also provide a good basis for the future HIV vaccine strategy.
There are only 18 representative whole genome sequences of HPV30 in the NCBI database. Variants of HPV30 have been classi ed as six phylogenic lineages based on the total sixteen whole genome sequences, these are lineage A1-A5 and B 6,8 . Lack of data on HPV30, on the one hand may be due to the low prevalence of HPV30 in women, on the other hand may be due to the limitations of detection technique. In early studies, HPV30 was mainly detected in the esophageal carcinogenesis and genital tract specimens by Southern Blot. In subsequent studies HPV30 detection was performed mostly by general primer PCR and direct sequencing of the PCR products.
Both the prevalence and lineage distribution of HPV30 are still nebulous. In this study, we screened and con rmed HPV30 infection among healthy women, to investigate the distribution of HPV30. Therefore, the prevalence of HPV30 was investigated by type speci c PCR among women with normal cervical cytology; lineage distribution of HPV30 was investigated based on the analysis of L2 DNA sequences; this study also analyzed the diversity of HPV30 genetic variants and the HPV30 proteins. This study may provide basic data for future studies on viral fundamental research and prevention.

Cervical specimens
In this study, totally 1600 specimens examined were obtained from outpatient women of the department of obstetric and gynecology at the Chinese PLA General Hospital in Beijing from January 2013 to July 2014. All the 1600 patients aged 15-89 years (median, 41 years) were diagnosed as normal cervical cytology. DNA DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
General primers PCR for HPV16 and HPV30 plasmids HPV30 plasmid was constructed using L1 PCR product (primer 9 and 10) of sample 9 cloned into pMD18-T vector. The HPV16 L1 plasmid was prepared in our laboratory before.
General PCR of PGMY09/11 9,10 , MY09/11 11,12 , PI 13 and FAP59/64 14 were done in a 20 µl reaction mix containing about 50 ng DNA, 4 mmol/L MgCl 2 , 100 µmol/L of each dNTP, 0.1 µmol/L of each oligonucleotide primer, and 1 U AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The temperature pro le used for ampli cation consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles with denaturation at 95°C for 20 s, annealing at 55°C for 2 min, and extension at 72°C for 1.5 min, which was extended for 5 min in the nal cycle. SKF/R 15 PCR were done in a 20 µl reaction mix containing about 50 ng DNA, 4 mmol/L MgCl 2 , 100 µmol/L of each dNTP, 0.1 µmol/L of each oligonucleotide primer, and 1 U ExTaq DNA polymerase (TaKaRa, Dalian). The temperature pro le consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles with denaturation at 98°C for 10 s, annealing at 45°C for 30 s, and extension at 72°C for 20 s, which was extended for 5 min in the nal cycle.
HPV30 detection with polymerase chain reaction (PCR) HPV30 type speci c PCR was performed with primer 1 and 2 (Table 1), with an amplicon size of 240 bp. Brie y, the reaction in a 50 µl reaction mix containing about 50 ng DNA, 2 mmol/L MgCl 2 , 100 µmol/L of each dNTP, 0.1 µmol/L of each oligonucleotide primer, and 1 U ExTaq DNA polymerase (TaKaRa, Dalian).
The temperature pro le consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles with denaturation at 95°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, which was extended for 5 min in the nal cycle. The PCR products were visualized on 1.5% ethidium bromide-stained agarose gel. Other HPV30 PCRs to obtain the full length of HPV30 genome(primer 3 and 4, primer 5 and 6, primer 7 and 8) were also done in a 50 µl reaction mix containing about 50 ng DNA, 4 mmol/L MgCl 2 , 100 µmol/L of each dNTP, 0.1 µmol/L of each oligonucleotide primer, and 1 U ExTaq DNA polymerase (TaKaRa, Dalian). The temperature pro le consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles with denaturation at 95°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 3 min, which was extended for 5 min in the nal cycle. The PCR products were visualized on 1.0% ethidium bromide-stained agarose gel and puri ed by using Qiagen gel extraction kit (Qiagen, Hilden, Germany

Results
Ampli cation e ciency of HPV30 comparison with general primer sets To assess the capacity of HPV30 detection, HPV16 was used as a control. Five sets of general primers targeting L1 gene including PGMY09/11, MY09/11, PI, FAP59/64 and SKF/R were used to detect HPV16 and HPV30 plasmids at the same time.
PGMY09/11 and SKF/R PCR were able to detect 1000 copies of HPV16 plasmid respectively; MY09/11, PI and FAP59/64 PCR were able to detect 100 copies of HPV16 plasmid respectively. PGMY09/11, MY09/11 and PI PCR were unable to detect HPV30 respectively, while FAP59/64 PCR was able to detect 100 copies of HPV30 plasmid, SKF/R PCR was able to detect 1000 copies of HPV30 plasmid. In summary, HPV16 could be detected by all the ve sets of primers, while HPV30 could be detected by the FAP59/64 and SKF/R (Fig. 1). Detection of HPV30 among women with normal cervical cytology HPV30 was screened in the 1600 cervical specimens from women with normal cytology by HPV30 type speci c PCR ( Fig. 2A). The positive control was a mixture of the 1600 samples. The identi cation was con rmed by the results of a single amplicon band of 240 bp, after screening, nine samples (numbers 1-9) showed the expected bands (Fig. 2B). Therefore, the infection rate of HPV30 was 0.56% (9/1600) in China.

Lineage distribution of HPV30 isolates
According to the previous HPV30 lineages studies, the whole genome sequences of HPV30 submitted to GenBank were separated into six distinct lineages A1, A2, A3, A4, A5 and B 6 .
The phylogenetic analysis was rstly conducted according to multiple nucleotide sequence alignments of whole genomes, including the six HPV30 genomes (sample 1, 3 and 6-9) identi ed in this study and other 18 known representative HPV30 variant lineages (for NCBI accession numbers see Fig. 3A).
Then the phylogenetic trees were constructed using the partial genome sequences of L2 DNA of the six HPV30 isolates identi ed in this study and the 18 NCBI downloaded HPV30 sequences. The classi cation result of L2 DNA was consistent with the result according to the full-length six isolates of HPV30 Finally, according to the phylogenetic tree of L2 DNA, among the nine HPV30 isolates (1-9) obtained in this study, six (sample 1, 4-6, and 8-9) belonged to lineage A1, two (sample 2 and 4) belonged to lineage A4, and the last one (sample 3) belonged to lineage A5, none belonged to lineage A2, A3 or lineage B (Fig. 3C).

Summary of the mutations of HPV30 isolates
Compared with the HPV30 prototype reference (GenBank: X74474), 87 variations in the nine HPV30 genomes identi ed in this study were observed (Table 3), including 79 substitutions, four insertions and four deletions. Among the six (sample 1, 3 and 6-9) which successfully retrieved HPV30 full-length sequences, the number of substitutions range from 12 to 39 (Table 2). Insertions and deletions were observed in E2, L2 and LCR, but not in other ORFs (E6, E7, E1, E5 and L1). The speci c mutation sites of each isolate were also shown.    (Fig. 4).
Of all these nine samples, the sample 5 had a C to T substitution (C25T) in the position of the ninth amino acid which led to the formation of the TGA stop codon (Fig. 5A). The sample 5 also had an insertion of C at the 493-497 position (a ve bp of CCCCC) of the original HPV30 reference (X74474) (Fig. 5B).

Discussion
Co-infection both with HIV and HPV as well is an important issue. Compared to HIV-negative women, HIV-infected women have a higher prevalence of HPV [14,[21][22][23] . Immune-suppression caused by HIV increases the persistence of HPV, and the risk for lesions and cancer in the cervix and other sites. As pregnancy is a unique immunological state, pregnancy is also associated with increased HPV prevalence, and with more rapid progression to intraepithelial lesion. HPV HPV30 is a relatively rarely reported genotype worldwide, with few data available to characterize its prevalence and lineage distribution, even in women with normal cervical cytology. This study speculates that the reason why HPV30 is less studied is due to the problem of HPV detecting. HPV typing is mostly based on the L1 sequences. The detection methods have mainly relied on PCR ampli cation by general primers which target the conserved regions of L1 gene of the virus genome, followed by HPV type identi cation of direct sequencing, or type-speci c oligonucleotide probes hybridization. However, these consensus primers have limitations, particularly in the variability in detection sensitivity among different HPV types. Taken an example, MY09/11 PCR was able to detect 10 copies of HPV 31, but the detection limit was 10 2 for HPV16, and 10 4 for HPV52 11 . As shown in Fig. 1, MY09/11 primer sets PCR lacked speci city for HPV30. We supposed that some HPV types like HPV30 could be missed in previous studies of which HPVs were detected by general primer sets. Nextgeneration sequencing (NGS) bypasses or reduces many problems due to PCR-based HPV detection approaches. By the use of NGS, the existence of HPV30 infection was also con rmed in lower genital tract of women 2,18,19 . The technical limitations may partially explain the reason why some HPV types like HPV30 are not detected in clinical specimen.
In this study, by using the HPV30 type speci c PCR targeting E6-E7 region (primer 1 and 2), the infection rate of HPV30 of 0.56% (9/1600) in China was determinated. Whole genome and partial HPV30 genome of these nine isolates were obtained, which veri es the reliability of the existence of HPV30 infection in these specimens. This infection rate is basically consistent with the 0.5% infection rate of Thailand 20 . Because of the samples collection, we did not get the samples with abnormal cervical cytology. But as the reference studies 20,21 , the abnormal group could get a higher infection rate of HPV30 than the normal group. In this study, the fragment deletion of HPV genome sequence in E2 or L2 genes was found in two different samples (E2 in sample 4, L2 in sample 5), which had not been reported in previous studies. The capsid protein of L2 is the minor capsid protein. L2 can be incorporated when co-expressed with L1 24 . L2 contains a major cross-neutralization epitope in the N-terminus and represents the target of neutralizing antibodies 23 . The sample 5 had an N-terminal truncated L2 (196 amino acids) compared with L2 of other isolates of HPV30 (463 amino acids). As the HPV L2 protein contains overlapping binding sites for neutralizing, cross-neutralizing and non-neutralizing antibodies among the N-terminal region 25 . The L2 of sample 5 could be a naturally defective L2 lack of the of N-terminal function. The sample 4 had a C-base deletion mutation occurred at position 767 of E2 DNA, resulting in the amino acid sequence shift after the position 256. And this frame shift could be the loss of E2 protein function of C-terminal DNA binding.

Conclusion
Our study showed that the infection rate of HPV30 is 0.56% in China; all of the HPV30 isolates belongs to lineage A. Natural L2 and E2 defectives isolate of HPV30 were found. As HPV30 could be an important HPV type in immunosuppressive women, sequence diversity and phylogenies of HPV30 provide basis for future researches on discrete viral, epidemiology, evolution, pathogenicity, and even the vaccines strategy.   Phylogenetic analysis of the L2 DNA of the HPV30 isolates. Phylogenetic analysis based on the Neighbor-Joining method of the relationships. Phylogenetic analyses were conducted in MEGA 6.0. A Sub-lineage classi cation was based on full genomes from a reported research. B The regions of L2 DNA sequence of the sixteen HPV30 isolates available at GenBank and six sequences (1, 3, 6-9) obtained in this study were analyzed. C The regions of L2 DNA sequence of the sixteen HPV30 isolates available at GenBank and nine sequences (1-9) obtained in this study were analyzed.