Molecular identification of novel viruses

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Molecular identification of novel viruses Paul Kellam

D

uring the past two decViruses are responsible for many of the identify new human viruses, ades, many new human diseases caused by microbial infection. such as hepatitis G virus (GBVviruses have been idenDuring the past two decades, ~20 new C)8, sin nombre virus (SNV)9 tified. Although some of these human viruses have been discovered. and HRV-5 (Ref. 4), and animal are not associated with known Many of these new viruses were initially viruses, such as pig endogendiseases, the majority cause identified using molecular biology ous retrovirus (PERV)10, walldevastating illnesses such as techniques, a major advantage of which is eye dermal sarcoma virus11 AIDS, hepatocellular carcinoma the ability to search rapidly for new and a macaque gamma herpesand haemorrhagic fevers1. viruses, known viruses or related, virus, retroperitoneal fibromaMany of these new viruses, for but previously unidentified, members tosis herpesvirus (RFHVMn)12. example hepatitis C virus of established virus families in However, the lack of specific (HCV)2, human herpesvirus 8 disease samples. controls for degenerate primers (HHV-8)3 and human retromeans standardization can only P. Kellam is in the Dept of Virology, virus 5 (HRV-5)4, have only be achieved for existing memInstitute of Cancer Research, been detected by using exbers of virus families, thereby Chester Beatty Laboratories, 237 Fulham Road, tremely powerful molecular not guaranteeing detection of an London, UK SW3 6JB. biology methods. New viruses unknown virus. The problems tel: +44 171 352 8133, fax: +44 171 352 3299, e-mail: [email protected] fall into the well-documented of inappropriate amplification field of emerging infectious disconditions can lead to the proeases and can be categorized as resurgent or recurrent duction of many false positives or, in some cases, no existing diseases, newly identified agents associated with amplification. Careful optimization of degenerate well-known human diseases, or new human diseases PCR conditions for virus family controls is therefore caused by pre-existing zoonotic agents (Box 1). The necessary. This usually requires optimization of reaction list of diseases with possible microbial aetiology is still buffers and primer annealing temperatures and the use large, and new pathogens are likely to be identified in of nested or semi-nested PCR strategies. In addition, some cases. Specific diseases, such as Kawasaki disease5, special amplification techniques, such as ‘touchdown sarcoidosis6 and multiple sclerosis (MS)7, as well as PCR’ can improve PCR amplification when degenerate broader disease groups, including neurodegenerative primers are used11,13. However, detection of virus disorders, forms of arthritis, inflammatory bowel dis- genomes in disease tissue does not automatically link a ease, autoimmune disease and certain cancers, might virus to a disease14, particularly when PCR screening is have an infectious origin. used to search for known or new viruses. For example, In the identification of an emerging virus, epidemio- several known infectious agents have been implicated logical data are often accompanied by clinical specimens in the pathogenesis of MS (Refs 7,15–17), but none of that have no reactivity with diagnostic reagents avail- these associations has been conclusively proved to able for known pathogens. The primary aim, therefore, cause MS (Ref. 18). is to identify any new infectious agent and to build a body of data to support the existence of a causal link Subtractive hybridization between microorganism and disease. In recent years, The most recent array of molecular techniques to be the emphasis of new virus discovery has shifted from adapted for use in new virus discovery is based solely the traditional methods of culture and serology to the on nucleic acids and makes no assumptions about the use of modern molecular biology techniques. nature of the virus agent present19. These methods have evolved from one aim of molecular medicine: to idenDegenerate PCR tify differences at the nucleic acid level between diseaseWith the widespread use of PCR and the availability associated tissue and normal tissue. The common feaof extensive sequence databases of virus genomes, it is ture of these methods is that one nucleic acid population often possible to design PCR primers that anneal to (‘uninfected’ or ‘driver’) is hybridized in excess with a conserved regions of virus genomes. These can then be second population (‘infected’ or ‘tester’) to remove comused to search for the presence of a given virus in new mon sequences, thereby enriching sequences unique to pathological specimens. The principles of degenerate the tester. In reality, the distinction between infected PCR are outlined in Box 2. Many research groups have and uninfected tissue is not always clear. Care must published degenerate PCR primers that recognize diverse be taken in choosing truly uninfected tissue, although virus families. These have been used successfully to quantitative differences in the amounts of infectious Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS

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Box 1. Categories of emerging virus diseases

Box 2. Degenerate PCR

Emerging infections can be broadly grouped into three categories:

Degenerate PCR primers contain mixed combinations of nucleotides at defined residues of the primer. These mixed bases correspond to regions of variation that occur between related viruses within a virus family. Mixed base (degenerate) oligonucleotides are synthesized by the reaction of equimolar amounts of the appropriate nucleotide with a synthetic DNA chain. For example, to make the sequence 5′..G(C or T)A..3′, an A residue is added, followed by equal amounts of C and T, followed by G. This produces a population of two distinct oligonucleotide primers (GCA and GTA). This primer population would therefore have a degeneracy of two. As more mixed bases are added, the degeneracy increases: for example, the primer 5′..T(C or T or A)G(C or T)A..3′ would be five nucleotides long and would consist of a population of six distinct oligonucleotides, TCGCA, TTGCA, TAGCA, TCGTA, TTGTA and TAGTA (a degeneracy of six). Degenerate PCR primers can be designed by comparing different virus genomes. Greater degeneracy can be obtained by designing primers based on conserved viral amino acid sequences following back-translation, rather than by simply comparing viral nucleic acids. Figure 1 illustrates these principles for the degenerate herpesvirus primer TGV (Ref. 36). Designing a complete PCR protocol with degenerate primers involves some strategic choices. The overall aim is to achieve a balance between covering all possible variants (i.e. primers with high degeneracy) and making the number of different primers unusably large. At high levels of degeneracy, only a very small proportion of the pool of primers are able to prime DNA synthesis, whereas a large proportion of the remaining primers will still be able to anneal but, because of sequence mismatches, will be refractory to PCR extension. A useful rule is to fix the level of degeneracy at ~256. Degeneracy can be reduced by using codon usage tables37 and intercodon dinucleotide frequencies38. However, in practice, the use of these tables for designing panviral family primers can be limited. For example, codon usage for isoleucine across seven herpesviruses shows considerable variation (Table 1). It is possible to reduce degeneracy further by using deoxyriboinosine as an alternative nucleotide in the PCR primer39. Overall degeneracy is less important at the 5′ end of the primer than at the discriminatory 3′ end. Finally, the 3′ nucleoside of the primer should not be degenerate.

Resurgent or recurrent diseases These diseases are usually caused by ‘new’ or mutated variants of previously known agents. A good example of this is the yearly appearance of new, antigenically different influenza viruses. These new variants are able to evoke disease in their host while causing the classical symptoms of influenza. New variants arise by two routes. Antigenic drift is caused by the accumulation of point mutations in the virus surface proteins. Antigenic shift is caused by the acquisition of a completely new coding sequence for one of the surface proteins of related influenza viruses from other animals, such as pigs or wildfowl. Newly identified agents associated with well-known human diseases The study of patients suffering post-transfusion hepatitis but with no evidence of infection by hepatitis A virus or hepatitis B virus led to the discovery of a new virus group, called the hepatitis C viruses (HCVs). HCVs are closely related to flaviviruses and pestiviruses. Serology assays have now shown that HCV is the major cause of non-A, non-B hepatitis throughout the world. New human diseases caused by pre-existing zoonotic agents HIV-1 and HIV-2, the aetiological agents of AIDS, have significant sequence homology to chimpanzee and simian immunodeficiency virus (SIV). AIDS is a new disease, and retrospective serological assays show that antibodies specific to HIV have only appeared in major human populations with the advent of the AIDS epidemic. This suggests that HIV and AIDS have resulted from a recent zoonosis from other primates.

nucleic acid between tester and driver are sufficient to allow the techniques to work. The methods can be broadly divided into physical subtraction techniques and PCR-based kinetic enrichment techniques. Each set of techniques has its own relative merits, which are ultimately dependent on the nature of the nucleic acid sample to be analysed. Physical subtraction techniques These methods are applicable only for detecting differences in mRNA expression between one cell type and another, but they can be used for detecting viral genomic RNA if it is polyadenylated. Early uses of this technique simply involved the solution hybridization of an excess of driver mRNA with cDNA made from tester mRNA. Common sequences present in both samples form cDNA–mRNA hybrids, leaving the unique sequences in the tester cDNA unhybridized. The doublestranded hybrids were removed by hydroxyapatite chromatography, exploiting the higher affinity of hydroxyapatite for double-stranded rather than singlestranded nucleic acids. The remaining subtracted cDNA was then used to construct a subtracted cDNA library or, if labelled, as an enriched cDNA probe for library screening. This method was used in combination with immunoscreening to identify the virus associated with

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Borna disease20,21, an infectious neurological disease that occurs sporadically in horses and sheep in central Europe and may also be associated with certain neuropsychiatric disorders in humans22. Modern versions of this technique use cDNA libraries constructed in the cloning vector λZap (Stratagene, La Jolla, CA, USA). The libraries are made from driver and tester cell lines or tissue samples (Fig. 2). This method has been used to isolate rare cDNAs (<0.01% abundance) from colon and hepatic carcinoma tissue23. The λZap vectors allow the rescue, directly from the library, of single-stranded DNA phagemids and DNA (ssDNA) or double-stranded DNA plasmids (dsDNA), all containing representative cDNAs. λZap vectors also allow the production of directionally cloned cDNAs. In such cDNA libraries, the 5′ cDNA ends are always associated with a T3 RNA polymerase promoter, and the 3′ cDNA ends are always associated with a T7 promoter, both of which are encoded in the λ phage multiple cloning site. Both primary cDNA libraries are amplified, and ~2 million plaque-forming-units (pfu) of the tester library are used to produce ssDNA. The same amount

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(a) Amino acid sequence Cys

Asn

Ala/Ser Phe/Val

Tyr

Gly

Phe/Val

Thr

Gly Val/Ala

Degeneracy

(b) Degenerate primer

TGY

AAY

KCB

KTB

TAY GGK

KTY

ACN GGV

GY

TGY

AAY

TCN or GCN

TTY or GTN

TAY GGN

TTY or GTN

ACN GGN

GTN or GCN

–

TGY

AAY

KCN

KTN

TAY GGN

KTN

ACN GGN

GY

131 072

TGT

AAC

TCG

GTG

TAY GGN

TTY

ACN

GT

256

55 296

based on nucleic acid similarity (c) Back-translation

of the amino acid sequence (d) Degenerate primer

based on backtranslated sequence (e) Final PCR primer

GGN

TGV Fig. 1. Design of degenerate PCR primers to the herpesvirus DNA polymerase. Based on sequence alignments of herpesvirus polymerase genes, a conserved region was identified at the (a) amino acid and (b) nucleic acid levels. The degeneracy of an oligonucleotide primer derived from nucleic acid alignments is 55 296. (c) Back-translation of the conserved amino acid sequence produces (d) a primer with a greater degeneracy of 131 072. Both primers are too degenerate to be useful. (e) By limiting the degeneracy to nucleosides adjacent to the terminal 3′ nucleotide, the final primer, TGV (Ref. 36), was designed with a degeneracy of 256. Key: Y = C or T; K = T or G; B = T, C or G; N = A, C, G or T.

(2 × 106 pfu) of driver library is used to produce dsDNA. In vitro, transcribed RNA incorporating dUTP-biotin is made from the driver dsDNA using a T3 promoter located in the vector. These biotinylated RNAs are complementary to the tester ssDNA and are subsequently hybridized to the tester ssDNA. The complementary hybrids are removed using streptavidin-coated beads, which bind to the biotin moiety of dUTP-biotin. When the beads are removed, the enriched tester cDNAs that did not hybridize remain in solution. Multiple rounds of hybridization and subtraction can be performed, at each stage enriching the concentration of rare cDNAs in the tester population. Following subtraction, the tester ssDNA is made double stranded and is transformed into Escherichia coli (Fig. 2). This represents the enriched subtracted library, which can then be analysed further.

adapted to identify differences in mRNA expression26 and has been successfully used to identify a new human virus associated with Kaposi’s sarcoma, namely HHV-8 (Ref. 3). RDA has also been used to identify the viruses GBV-A and GBV-B (Ref. 27), which are related to GBV-C (Refs 8,28). More controversially, RDA has been used to identify HHV-6 sequences in plaques from patients with MS (Ref. 16) and cDNA clones from infectious Creutzfeldt–Jakob brain fractions that have no homology to any known database sequences29. RDA combines three elements: representation, subtractive enrichment and kinetic enrichment. The procedure is carried out in two stages. The first stage comprises the preparation of ‘representations’ for driver and tester DNAs or cDNAs. These are basically small restriction enzyme fragments that ‘represent’ the total tester and driver nucleic acids. Restriction fragments, deRepresentational difference analysis (RDA) rived from the starting nucleic acid, are ligated to oligoRDA represents the first global approach to analyse nucleotide adapters and amplified by PCR. The second differences between cellular genomes. Although orig- stage consists of reiterative hybridization/selection inally developed to look for differences between tumour steps (Fig. 3a). Before hybridization, the oligonucleotide cell genomic DNA and normal cells24,25, it has been adapters used for the initial representation PCR step are cleaved from the driver and tester PCR products (amplicons), and a new set of defined but different sequence 37,a,b Table 1. Isoleucine codon frequency (frequency per 1000 codons) adapters are ligated onto the 5′ ends of the tester amplicons. After hybridc HSV-1 HSV-2 EBV CMV HHV-6 EHV-1 HVS Human ization of tester and driver amplicons, the mixture of molecules is treated AUA 3.4 3.4 6.8 4.9 21.4 14.1 27.7 6.1 with DNA polymerase, which adds a AUC 21.5 27.4 17.4 26.3 24.0 17.0 7.3 24.3 AUU 5.3 3.6 11.8 9.9 28.0 11.8 32.0 15.0 copy of the defined oligonucleotide to both 3′ ends of the self-annealed a Numbers in bold represent the most commonly used isoleucine codon in each case. tester DNA fragments only. The deb Abbreviations: CMV, cytomegalovirus; EBV, Epstein–Barr virus; EHV, equine herpesvirus; HHV, fined oligonucleotide adapter/primer human herpesvirus; HSV, herpes simplex virus; HVS, herpesvirus saimiri. c is then used during PCR of the mixDetermined from the coding sequence of random human cDNAs. ture, such that only the tester-annealed

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DNA fragments can participate in exponential amplification to produce a difference product. The cycle of (1) cleavage of old adapters and ligation of new adapters to the difference products, (2) hybridization with excess driver and (3) PCR amplification is repeated two to three times, and the final PCR products are cloned and analysed further.

(a)

Infected (tester) cell λZap cDNA library

Uninfected (driver) cell λZap cDNA library

dsDNA Suppression subtraction hybridization (b) rescue (SSH) SSH, a technique based on similar principles to RDA, was described in 1996 (Ref. 30) and is designed to selectively amplify differentially exIn vitropressed cDNA fragments and simulsynthesized ssDNA taneously suppress non-targeted DNA biotinylated rescue amplification. This method has not RNA yet been used for virus identification but, like RDA, has enormous potential. Analogous to RDA, it can be used (c) Hybridize 1:10 on a variety of nucleic acid targets (Fig. 3b), and representations of both tester and driver DNAs are prepared by restriction enzyme digestion. Tester DNA is then subdivided into two portions and each is ligated with a different oligonucleotide adapter to the 5′ end. However, in contrast to RDA, SSH involves two hybridization steps. In the first, an excess of denatured Removal of driver cDNA is added to each popu(d) biotinylated lation of denatured tester cDNA. hybrids using Owing to the second-order reaction avidin agarose kinetics of hybridization, singlestranded molecules corresponding to high- and low-abundance sequences Convert ssDNA to dsDNA become normalized. Normalization and transform bacteria occurs because the annealing process is faster for more-abundant molecules and results in a propor(e) tion of low-abundance tester cDNA remaining single stranded. During Screen for differentially the second hybridization, the two expressed mRNA primary hybrid samples are combined. and analyse As the samples are not heat denatured, only the remaining normalized and Fig. 2. Schematic representation of subtractive cDNA libraries, based on the method of Schweinfest subtracted single-strand tester DNAs et al.23 (a) Two cDNA libraries, one from infected cells and one from uninfected cells, are constructed in the vector λZap. (b) Single-strand phagemid DNA (ssDNA) is rescued from ~2 × 106 are able to associate to form new hyplaque-forming units (pfu) of the infected library, and double-strand phagemid DNA (dsDNA) is brids. These hybrids have different rescued from the same amount of uninfected library. (c) Biotinylated RNA is synthesized in vitro oligonucleotide adapter sequences at from the dsDNA and is then hybridized with the ssDNA. (d) DNA–RNA hybrids are removed by their 5′ ends. Fresh denatured driver avidin agarose affinity beads, and the remaining ‘subtracted’ ssDNA is converted into dsDNA and (e) transformed into Escherichia coli. DNA is then added to further enrich for the differentially expressed sequences. Following hybridization and DNA polymerase end-filling, the entire population is minally repeated primer sites30. Only hybrids with difPCR amplified with oligonucleotides for both adapt- ferent adapters at either end are exponentially amers. Hybrids with the same adapter at either end are plified. A final nested PCR with internal common suppressed from PCR amplification because of the primers allows the cloning and analysis of the difference formation of pan-handle structures between the ter- products.

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(a)

(b)

Driver amplicon

Tester amplicon

Tester with adapter 1

Ligate to primer

Driver (in excess)

Tester with adapter 2

First hybridization i ii

Mix, denature, anneal

iii iv

ds-tester

Hybrids

Digest with restriction endonuclease

ss-tester

ds-driver

{

ss-driver

Second hybridization: mix samples, add fresh denatured driver, and anneal

Fill in ends i, ii, iii, iv + v

Fill in ends i ii

Add primer, PCR amplify

iii iv

Linear amplification

{

v Exponential amplification

No amplification Add primers, PCR amplify Digest ssDNA with mung bean nuclease, PCR amplify Difference product enriched in target

Clone and analyse

i, iii

Linear amplification

ii

No amplification

iv

No amplification

v

Exponential amplification

Difference product Clone and analyse Fig. 3. Schematic representation of different sequence-based PCR methods used to identify unculturable infectious agents. (a) Representation difference analysis (RDA). Modified with permission from Ref. 24. (b) Suppression subtraction hybridization (SSH). Modified with permission from Ref. 30. Purple and green boxes represent DNA or cDNA, and yellow, white and red boxes represent oligonucleotide primers/adapters. With both methods, representations of tester (purple boxes) and driver (green boxes) DNAs are produced by restriction enzyme digestion. (a) For RDA, specific oligonucleotide adapters are ligated onto the tester DNA only. The tester and driver DNAs are then mixed, denatured and annealed to produce a mixed population of double-strand (dsDNA) and single-strand (ssDNA) molecules. Following DNA polymerase end-filling and removal of ssDNA with mung bean nuclease, the mixture is PCR-amplified with a primer specific for the adapter that was originally ligated onto the tester DNA. This enables the exponential amplification and, therefore, enrichment of only the re-annealed tester DNA. This process is then repeated multiple times. (b) For SSH, following restriction enzyme digestion, the tester DNA is divided into two portions onto which two different oligonucleotide adapter sequences are ligated (white/yellow boxes and white/red boxes). Each tester sample is mixed with driver, denatured and allowed to hybridize, producing a population of different re-annealed molecules (i–iv). A second hybridization is performed by mixing the first two samples and adding fresh denatured driver DNA. This enables the formation of another form of tester dsDNA molecule (v) that has different adapters at the ends. Following DNA polymerase end-filling, the mixture is PCR-amplified with primers specific for both adapters. Exponential amplification only occurs for molecules with both adapters present (v).

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New virus discovery The correct combination of methods needs to be used for the variety of samples that are likely to arise in new virus discovery. If the biological samples available are cell-free, the choice of methods is limited: with no knowledge of the nature of the viral genome (i.e. DNA, positive-sense RNA or negative-sense RNA), degenerate PCR, RDA or SSH is the method of choice. However, for infected cell lines or tissue samples, all methods are applicable. If the nature of a new viral genome is not known, the most global method of analysis would be to study new mRNA species. The use of physical subtraction methods requires a relatively abundant supply of poly(A)+ mRNA, which may limit the technique. Another disadvantage is the requirement for cDNA libraries, which are costly and time-consuming to make. However, as these methods avoid the use of PCR, and thus PCR contamination, they are still very useful. RDA and SSH also have their disadvantages. They require complex restriction digests, primer ligations, hybridization and PCR, and contamination with common laboratory DNA at the initial stages can lead to false positive clones. In addition, failure at any one stage can lead to a lack of difference PCR products or false positives. RDA is more time-consuming than SSH and also requires more initial tester and driver nucleic acid. Following the identification of a candidate clone, further analysis to characterize whether the candidate is truly exogenous in origin is required. Analysis of open reading frames should show if the clone codes for any known homologous protein and whether any such proteins are related to known viruses. Advances in technology over the last ~70 years have brought new problems in assigning causation to newly identified pathogens and have re-emphasized the limitations of the original Henle–Koch postulates. The new concepts and technology involved in proving causation of disease have been extensively reviewed by Alfred Evans in the formulation of his proposed ‘elements of immunologic proof of causation’31. The ability to culture viruses in vitro and the detection of antibodies raised against viruses were two major advances in technology and understanding that led to the new proposals. For example, immunological data were used to show that Epstein–Barr virus (EBV) was indeed the cause of infectious mononucleosis32. Subsequently, the role of EBV in causing Burkitt’s lymphoma and nasopharyngeal carcinoma was established. However, even with the new criteria, errors occur. For example, similar serological and virological evidence strongly implicated herpes simplex virus type 2 as the cause of cervical cancer, but it was not until the discovery of human papilloma virus DNA within cervical cancer biopsies that the major cause of cervical cancer was uncovered33. Sequence-based approaches for new virus identification can also lead to misleading conclusions when viewed in isolation. This has led to the formulation of revised guidelines for defining the causal relationship between the presence of viral sequences and a disease14. In the absence of other corroborating data, the presence of viral sequences within disease tissue is insufficient to prove that a new virus causes a particular disease.

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Questions for future research • How many new human pathogens remain? Are we just seeing the tip of an iceberg? • Do the principles of causation require further refinement? • Will genomics-based research strategies help reveal new pathogens? • Is there sufficient vigilance to identify and prevent another AIDSlike epidemic?

Detection of viruses may reflect either the presence of virus in surrounding cells and tissues or the ability of a virus to replicate within the microenvironment of the disease tissue, rather than the virus causing the observed disease. Epidemiological, immunological and sequence-based criteria should be combined to demonstrate causality. These were used effectively to link HCV sequences causally with the clinical condition of nonA, non-B hepatitis34, and are currently being employed to determine the link between HHV-8 and Kaposi’s sarcoma. The use of existing criteria for causality is likely to be rigorously tested in attempts to define the role of new aetiological agents when they constitute a single component of a disease of multifactorial origin. Disease may result from multiple exposures to a virus, as is the case for dengue fever, or may arise only after long-term chronic asymptomatic infections. Chronic persistent infection combined with a genetic predisposition in the host may also account for some diseases. In trying to elucidate the mechanism of complex diseases, determination of the genetic component in the absence of an infectious component could lead to an incomplete understanding of the disease. The ingenuity of molecular biological approaches should help unravel the causes of many pre-existing diseases, at the same time enabling the rapid identification of any new, virulent, emerging infectious disease. However, as noted in 1937 by the distinguished American virologist Thomas Rivers, commenting on the ingenuity of the investigator, ‘to obtain the best results, however, this ingenuity must be tempered by the priceless attributes of common sense, proper training, and sound reasoning’35. Acknowledgements I thank Robin Weiss and David Griffiths for their helpful suggestions and comments on this paper. References 1 Satcher, D. (1995) Emerg. Infect. Dis. 1, 1–6 2 Choo, Q.L. et al. (1989) Science 244, 359–362 3 Chang, Y. et al. (1994) Science 266, 1865–1869 4 Griffiths, D.J. et al. (1997) J. Virol. 71, 2866–2872 5 Kawasaki, T. et al. (1974) Pediatrics 54, 271–276 6 Newman, L.S., Rose, C.S. and Maier, L.A. (1997) New Engl. J. Med. 336, 1224–1234 7 Allen, I. and Brankin, B. (1993) J. Neuropathol. Exp. Neurol. 52, 95–105 8 Simons, J.N. et al. (1995) Nat. Med. 1, 564–569 9 Nichol, S.T. et al. (1993) Science 262, 914–917 10 Patience, C., Takeuchi, Y. and Weiss, R.A. (1997) Nat. Med. 3, 282–286

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Zhang, Z. and Martineau, D. (1996) J. Virol. Methods 60, 29–37 Rose, T.M. et al. (1997) J. Virol. 71, 4138–4144 Don, R.H. et al. (1991) Nucleic Acids Res. 19, 4008 Fredericks, D.N. and Relman, D.A. (1996) Clin. Microbiol. Rev. 9, 18–33 Kurtzke, J.F. (1993) Clin. Microbiol. Rev. 6, 382–427 Challoner, P.B. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7440–7444 Perron, H. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7583–7588 Rice, G.P. (1992) Curr. Opin. Neurol. Neurosurg. 5, 188–194 Gao, S.J. and Moore, P.S. (1996) Emerg. Infect. Dis. 2, 159–167 Lipkin, W.I. et al. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4184–4188 VandeWoude, S. et al. (1990) Science 250, 1278–1281 Bode, L. et al. (1995) Nat. Med. 1, 232–236 Schweinfest, C.W. et al. (1990) Genet. Anal. Tech. Appl. 7, 64–70 Lisitsyn, N. and Wigler, M. (1995) Methods Enzymol. 254, 291–304 Lisitsyn, N., Lisitsyn, N. and Wigler, M. (1993) Science 259, 946–951

Everything a GP should know Infection (Oxford General Practice Series) by L. Southgate et al. Oxford University Press, 1997. £27.50 pbk (viii + 468 pages) ISBN 0 19 262092 4

I

nfection is the latest edition to a series of books designed for general practitioners (GPs). Not being a GP myself, I have not had the pleasure of reading any of the other books in this series, but if Infection is anything to go by, they must be valued very highly by their readership. I do not feel completely unqualified to review this book, however, having more than one GP in my immediate family! The book is organized into infections of the various systems of the

How to treat a pathogen Medical Microbiology (3rd edn) by P.R. Murray et al. Mosby, 1997. $54.00 pbk (x + 719 pages) ISBN 0 8151 9035 2

T

his is the third edition of a standard American textbook on medical microbiology. Like its previous editions, it is

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26 Hubank, M. and Schatz, D.G. (1994) Nucleic Acids Res. 22, 5640–5648 27 Simons, J.N. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3401–3405 28 Linnen, J. et al. (1996) Science 271, 505–508 29 Dron, M. and Manuelidis, L. (1996) J. Neurovirol. 2, 240–248 30 Diatchenko, L. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6025–6030 31 Evans, A.S. (1976) Yale J. Biol. Med. 49, 175–195 32 Henle, G., Henle, W. and Diehl, V. (1968) Proc. Natl. Acad. Sci. U. S. A. 59, 94–101 33 Durst, M. et al. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3812–3815 34 Kuo, G. et al. (1989) Science 244, 362–364 35 Rivers, T.M. (1937) J. Bacteriol. 33, 1–12 36 VanDevanter, D.R. et al. (1996) J. Clin. Microbiol. 34, 1666–1671 37 Wada, K. et al. (1992) Nucleic Acids Res. 11, 2111–2118 38 Smith, T.F., Waterman, M.S. and Sadler, J.R. (1983) Nucleic Acids Res. 11, 2205–2220 39 Martin, F.H. et al. (1985) Nucleic Acids Res. 13, 8927–8938

body, with other sections on general principles, such as the prevention of infections, the response of the body to infection, investigation and treatment. It concludes with several chapters on generalized infections, infection during travel abroad, and HIV. Each chapter is well referenced, with further reading for those with a greater depth of interest, thus providing a more than adequate source of knowledge for even the most conscientious GP. As a scientist/clinician interested in the field of respiratory infection, I felt that there were one or two shortcomings in the chapters dealing with this subject; in particular, I felt that the role of virus infections in sore throat and otitis media was rather underemphasized and that insufficient attention was paid to the problem of overprescribing of antibiotics for viral infections. Despite being the cause of the major-

ity of upper respiratory tract infections, rhinoviruses hardly featured at all, and there was disappointingly little on the newer macrolide antibiotics and on the spectrum of activity of new classes of antibiotics, such as quinolones (for example, ciprofloxacin). Notwithstanding these criticisms, these chapters, and the book as a whole, provide a very informative, educated and well-reasoned approach to an important subject. Infection will be of interest to medical students, GPs, and those advising GPs on the treatment of infection, although its enjoyable style of writing also lends itself to the lessexperienced reader.

beautifully produced with colour line drawings and is lucidly written by experts. The format is unchanged from previous editions, with sections on microbial physiology, pathogenesis, chemotherapy and systematic treatises on the pathogens. There are also case studies and questions at the end of each chapter, which have the dual purpose of putting the text into context for the trainee physician and testing knowledge

retention. In general, the book is up to date; for example, there is a discussion on combination therapy for HIV. However, in some chapters, the lack of references from the past 10 years is surprising because there have been so many major advances in medical microbiology. In addition, I am in favour of discontinuing the term ‘slow viruses’ for prions; although some doubt exists about the latter,

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Sebastian L. Johnston University Medicine, Southampton General Hospital, Tremona Road, Southampton, UK SO16 6YD

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