1202

Selective Detection and Differentiation of Subgenus B

Adenoviruses (Types 3, 7 and 11) by

Polymerase Chain Reaction

Tsuguto FUJIMOTO1), Masatsugu CHIKAHIRA1) and Osamu NISHIO2)1)Division of Microbiology, Hyogo Prefectural Institute of Public Health

2)National Institute of Public Health

(Received: June 4, 1998)(Accepted: August 19, 1998)

Key words: adenovirus, PCR, hexon, subgenus B

Summary

Application of the polymerase chain reaction (PCR) method for detection of subgenus B adenovi-

ruses (types 3, 7 and 11) was investigated. It is based on a simple (nonnested) PCR using primer pairs

specific for the hexon-coding region. The PCR allowed amplification of DNA from subgenus B

adenovirus prototype strains (types 3, 7 and 11) and adenovirus isolates (types 3 and 7), whereas it did

not amplify DNA from subgenus A (type 31), C (types 1, 2, 5 and 6), D (types 8, 19 and 37), E (type 4)

and adenovirus isolates (types 1, 2, 5 and 6). These results suggest that subgenus B adenoviruses

(types 3, 7 and 11) are detectable selectively by means of PCR with primer pairs developed in this

study. Amplified fragments from adenovirus types 3, 7 and 11 could be differentiated with restriction

endonuclease analysis with Rsa I.

Introduction

Human adenoviruses constitute a large family with 49 serotypes identified so far. These sero-

types are classified into six subgenera (A to F) on the basis of biochemical, immunological, and

morphological criteria1,2). Subgenus B of adenoviruses has been divided into two DNA homology

groups: B1 and B23). Both groups are associated with occasional severe infections and fatalities4,5). In

1996, adenovirus type 7 was isolated from fatal pneumonic cases in children with basal ailments in the

heart or lung, showing a new aspect of emerging infectious diseases in Japan4).

In the serotyping of subgenus B adenovirus isolates by the neutralizing test, attention should be

paid to a possible cross-reaction arising between type 7 and types 3 or 114). Recently, PCR has been

applied successfully in adenovirus detection in clinical specimens6)•`8). In this paper, we describe a new

PCR method for selective detection of subgenus B adenoviruses (types 3, 7 and 11). This rapid method

facilitates identification of these adenoviruses and may be useful for epidemiological surveillance.

Materials and Methods

1. Prototype strains

Prototype strains of human adenovirus types 1-8, 11, 12, 19, 31 and 37 were obtained from

American Type Culture Collection (Rockville, MD USA) or National Institute of Infectious Diseases

(Tokyo, Japan).

Correspondence to: Tsuguto FUJIMOTO

Division of Microbiology, Hyogo Prefectural Institute of Public Health, 2-1-29, Arata-cho, Hyogo-ku, Kobe

652-0032, Japan

感染症学雑誌 第72巻 第11号

PCR for Diagnosis of Subgenus B Adenoviruses 1203

2. Isolation and serotyping of adenoviruses from clinical specimens

Adenovirus isolates were obtained by inoculation of clinical specimens (nasopharyngeal swabs

and feces), which had been collected for surveying etiological agents in Hyogo Prefecture, onto 80%

confluent monolayers of Hep-2 cells in duplicated wells of a 24-well plate (Nippon Becton Dickinson,

Tokyo, Japan). When a cytopathic effect was evident, the isolates were serotyped by neutralization

assay9) using antisera obtained from National Institute of Infectious Diseases (Tokyo, Japan) or

Denkaseiken (Tokyo, Japan).

3. Viral DNA preparation

Viral DNA was prepared by a modification of the method described by Shinagawa et al.10)

Briefly, Hep-2 cell cultures were prepared in 25cm2 tissue culture flasks (Nippon Becton Dickinson,

Tokyo, Japan) and infected with adenoviruses. When most cells showed cytopathic effect, the culture

was removed and washed twice with PBS (-). The cells were lysed with 1ml of 0.6% SDS/10mM

EDTA and transferred into a 1.5ml centrifuge tube. Most of cellular DNA was removed as

precipitate by adding 0.3ml of 5 M NaCl, mixing well, keeping on ice for 3 hr and centrifuging at

15,000 rpm for 15min. The supernatant was transferred into 2 ml centrifuge tube and incubated with

200ƒÊg of proteinase K (GIBCO BRL, Rockville, MD USA) at 37•Ž for 1hr. The proteinase K treated

sample was extracted twice with an equal volume of phenol/chloroform mixture. After precipitation

with one volume of isopropanol, DNA was resolved in 50ƒÊl of distilled water.

4. Primers and PCR amplification

Figure 1 shows the oligonucleotides used as primers for PCR amplication. Primer pairs, designat-

ed as Hyogo 1997 U and Hyogo 1997, were newly designed on the basis of hexon sequencing of

adenovirus types 32) and 711). The other primer pairs (HexAA 1885 and HexAA 1913) were the general

ones described by Allard et al.7). Both of these primer pairs generate 301-bp PCR products. All

primers were synthesized by GIBCO BRL (Rockville, MD USA).

An aliquot (1ƒÊl) of the appropriately diluted DNA preparation was used as the template.

Amplification reactions were conducted in 50ƒÊl of reaction mixture containing 0.5ƒÊM each of the

primer pairs, 200ƒÊM each dideoxynucleotide, 1.25 U Taq polymerase (TaKaRa Shuzo, Shiga, Japan),

Fig. 1 Oligonucleotide sequences used in this study. Hyogo 1997 U and Hyogo

1997 are newly synthesized and used as primer pairs with PCR A. HexAA 1885

and HexAA 1913 are used as primer pairs with PCR B. The sequences are

shown to illustrate their homology. Nucleotides that are identical to the

preceding nucleotides sequence are marked by asterisks (*).

Sence primer

Antisense primer

+The numbering refers to the nucleotides' positions based on

those of Adenovirus type 2

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1204 Tsuguto FUJIMOTO et al

10mM Tris-HCl, pH 8.3,50mM KCl and 1.5mM MgCl2. The PCR was carried out using a cycle of

94•Ž for 30 seconds, 60•Ž for 30 seconds and 75•Ž for 30 seconds, and was continued for 30 cycles; in

the first cycle, the denaturation step continued for 7 minutes at 94•Ž, and in the last cycle, the

extension step continued for 5 minutes at 75•Ž. PCR using the newly synthesized primer pairs (PCR

A) and PCR using the general primers7) (PCR B) were performed in parallel. Five ƒÊl of the final

reaction product was analyzed in a regular 1.5% agarose gel and stained with etidium bromide.

5. Restriction enzyme analysis of PCR products

Aliquots of 10ƒÊl of PCR A reaction amplimer were digested for 6 hr with 10 U of Rsa I under

conditions specified by its manufacturer (New England Biolabs, Beverly, MA USA). Enzyme digests

were loaded onto 12% polyacrylamide gel and stained with etidium bromide.

Results

1. Optimizing the PCR

The newly developed PCR for subgenus B adenoviruses was tested with DNA from prototype

adenovirus type 3 using different Mg2+ concentrations (0.5-4.5mM). The bands of 301-bp were

clearly visible with 1.5mM concentration of Mg2+ (Fig. 2 PCR A). However, the general primers

tested under the same conditions did not amplify DNA fragments when Mg2+ concentration was less

than 2mM (Fig. 2 PCR B). From these preliminary experiments, 1.5mM MgCl2 was used thereafter.

Fig. 2 Effect of Mg2+ on PCR. DNA from prototype strain of adenovirus type 3 was am-

plified, using Mg2+ concentrations ranging from 0.5 to 4.5mM (left to right, with 0.5mM

increments; lane 1-9). PCR A and PCR B were performed parallelly using primers (Hyogo

1997 U and Hyogo 1997) and primers (HexAA 1815 and HexAA 1913) respectively. L; 100 bp

DNA Ladder (New England Biolabs, Bevery, MA USA).

PCR A

PCR B

感染症学雑誌 第72巻 第11号

PCR for Diagnosis of Subgenus B Adenoviruses 1205

2. Selective detection of subgenus B adenoviruses by the newly developed PCR

The two PCR methods were tested for adenoviruses prototype strains (types 1-7, 11, 19, 31 and

37). PCR A amplified 301-bp fragment with adenovirus types 3, 7 and 11. However, DNA from

adenovirus types 1, 2, 4, 5, 6, 19, 31 and 37 (non-subgenus B adenoviruses) did not amplify by the PCR

A. In contrast, the PCR B amplified DNA from non-subgenus B adenoviruses. Therefore, selectivedetection of subgenus B adenoviruses (types 3, 7 and 11) was possible by means of the PCR A with

primer pairs developed in this study (Fig. 3).DNA from clinically isolated adenoviruses were examined by the PCRs A and B. All of the DNA

from adenovirus strains serotyped 3 or 7 was selectively detected by the PCR A. The PCR B amplified

301-bp fragment DNA from adenoviruses typed 1, 2, 5 and 6 (Table 1).

3. Restriction enzyme analysis of PCR products generated by PCR A

Restriction enzyme analysis was performed using amplimers obtained with PCR A. Differentia-

tion was possible based on specific patterns (types 3, 7 and 11) in electrophoresis gel after treatmentwith Rsa I (Fig. 4 and Fig. 5).

Discussion

In this paper, we described a new PCR method, PcR A, to detect selectively adenoviruses types

3, 7 and 11. The method amplifies the same region of the hexon gene identified as group-specific byAllard et al.7). They reported primer pairs (HexAA 1885 and HexAA 1913) that anneal to the

group-specific region of adenoviruses. They detected 18 of 18 adenovirus types representing all sixsubgenera by PCR7). Their method is very simple and useful but the 3' end of their downstream primer

(HexAA 1913) does not exhibit precise base pairing with hexon-coding regions of adenovirus types 3

Fig. 3 Agarose gel electrophoresis of PCR amplicons with prototype strains of adenoviruses.Adenovirus types 1, 2, 3, 4, 5, 6, 7, 8, 11, 19, 31 and 37 are shown left to right. N; negativecontrol. L; 100 bp DNA Ladder (New England Biolabs, Bevery, MA USA).

PCR A

PCR B

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1206 Tsuguto FUJIMOTO et al

Table 1 Comparison of PCR A (with Hyogo 1997 U and Hyogo 1997)

and PCR B (with HexAA 1885 and HexAA 1913) for clinical isolates in

Hyogo Prefecture.

Fig. 4 Comparison of Rsa I restriction patterns of

amplified DNA of adenovirus prototypes 3, 7 and

11. L: 100 bp DNA Ladder.

Fig.5 Schematic presentation of Rsa I restriction

patterns of PCR products(adenovirus types 3 and

7).

*p; prototype strain

*i; clinically isolated strain

and 7 at their 3' ends. Although, the terminal match at the 3' end is critical for PCR amplification,

a mismatched T at the 3' is known to allow amplification if the PCR is proformed in non-stringent

conditions12). In this study, their primers did not allow amplification of DNA from adenovirus type 3

with Mg2+ concentrations less than 2mM. Morris et al. evaluated the same primers (Hex 1885 and

HexAA 1913) on ocular specimens and reported that DNA prepared from adenovirus type 7 gave no

amplified product with an annealing temperature of above 55•Ž8).

We sequenced 2 and 8 strains of adenovirus type 3 and 7 , respectively, which had been sampled

in Japan, Nepal and Indonesia, and confirmed that newly synthesized primer pairs (Hyogo 1997 U and

Hyogo 1997) exhibit precise base paring with target DNA of these isolates (unpublished data) . Thus,

PCR A was very sensitive to the detection of these two serotypes .

Amplimers of PCR A could be differentiated by endonuclease (Rsa I) analysis . Kase et al.13)

described that Rsa I was capable of discriminating PCR (they used primers by Allard et al .7) products

of adenovirus types 3 and 7. In this study, we also found that adenovirus type 11 was distinguishable

from types 3 and 7.

Further investigation must be done with adenovirus types 14, 16 , 21, 34 and 35 (other subgenus B

adenoviruses) to clarify whether these viruses can be detected and differentiated by our newly

developed PCR method.

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PCR for Diagnosis of Subgenus B Adenoviruses 1207

Acknowledgments

This study was partly supported by the Fund of Kidney Disease Research from Hyogo Total

Health Association.

References

1) Green M, Mackey JK, Wold WSM et al.: Thirty-one human adenovirus serotypes (Adl-Ad31) form five groups(A-E) based upon DNA homologies. Virology 1979; 93: 481-492.

2) Pring-Akerblom P, Trijssenaar FEJ and Adrian T: Sequence characterization and comparison of human adenovi-rus subgenus B and E hexons: Virology 1995; 212: 232-236.

3) Li Q-G, Hambraeus J and Wadell G: Genetic relationship between thirteen genome types of adenovirus 11,34, and35 with different tropisms: Intervirology 1991; 32: 338-350.

4) Infectious Disease Surveillance Center: Adenovirus type 7, Japan, April 1995-December 1996. IASR 1997; 79-80.5) Stalder H, Hierholzer JC and Oxman MN: New human adenovirus (candidate adenovirus type 35) causing fatal

disseminated infection in a renal transplant recipient: J Clin Microbiol 1997; 6: 257-265.6) Hierholzer JC, Halonen PE, Dahlen PO et al.: Detection of adenovirus in clinical specimens by polymerase chain

reaction and liquid-phase hybridization quantitated by time-resolved fluorometry: J Clin Microbiol 1993; 31:1886-1891.

7) Allard A, Girones R and Wadell G: Polymerase chain reaction for detection of adenoviruses in stool samples: J ClinMicrobiol 1990; 28: 2659-2667.

8) Morris DJ, Bailey AS, Cooper RJ et al.: Polymerase chain reaction for rapid detection of ocular adenovirusinfection: J Med Virol 1995; 46: 126-132.

9) Fife KH, Ashley R, Shields AF et al.: Comparison of neutralization and DNA restriction enzyme methods fortyping clinical isolates of human adenovirus: J Clin Microbiol 1985; 22: 95-100.

10) Shinagawa M, Matsuda A, Ishiyama T et al.: A rapid and simple method for preparation of adenovirus DNA frominfected cells: Microbiol Immunol 1983; 27: 817-822.

11) Pring-Akerblom P, Trijssenaar FEJ and Adrian T: Hexon sequence of adenovirus type 7 and comparison withother serotypes of subgenus B: Res Virol 1995; 146: 383-388.

12) Mahbubani MH and Bej AK: Applications of polymerase chain reaction methodology in clinical methodology inclinical diagnostics. In: Griffin HG and Griffin MG ed, PCR technology current innovation, CRC Press, Boca Raton,1994; 307-326.

13) Kase T, Okamoto S, Maeda A et al.: Application of the polymerase chain reaction assay in diagnosing adenoviruskeratoconjunctivities: J Eye (Atarashii Ganka) 1993; 10: 91-95.

PCRお よび制 限酵 素解 析 に よるア デ ノウ イル スB亜 群(3,7お よび11型)の

選 択 的 な検 出お よび区別 法

1)兵庫県立衛生研究所,2)国立公衆衛生院

藤本 嗣人1)近 平 雅嗣1)西 尾 治

要 旨

ア デ ノ ウ イ ル スB亜 群(3,7お よ び11型)を

他 亜 群 ア デ ノ ウ イ ル ス か ら区 別 す るPCR法 に つ

い て検 討 し た.こ の 方 法 は ヘ キ ソ ン コ ー デ ィ ン グ

領 域 に特 異 的 な プ ラ イ マ ー対 を用 い たPCR(入 れ

子PCRで な い)に よ る.同PCRに よ りB亜 群 標

準 株(3,7お よ び11型)お よ び ア デ ノ ウ イル ス

分 離 株(3お よ び7型)のDNAは 増 幅 した が,A

亜 群(31型),C(1,2,5お よび6型),D(8,

19お よび37型),E(4型)お よびアデノウイルス

分離株(1,2,5お よび6型)か らのDNAは 増

幅しなかった.こ れ らの結果よにり,こ の研究で

開発 したプライマー対 を使用したPCR法 により

アデノウイルスB亜 群(3,7お よび11型)が 選

択的に検出可能であることが示唆された.ア デノ

アイルス3,7お よび11型 の増幅産物 はRsa Iに

よる制限酵素解析により区別可能であった.

平成10年11月20日