Jumat, 26 Agustus 2011

VIROLOGICAL COMPLIANCE

7.1 Introduction

7.2 Health significance of human viruses in drinking-water

7.3 Occurrence of human viruses in source waters

7.4 Risk management

7.4.1 International approaches

7.4.2 Virus removal by current water treatment processes

7.5 Sampling, testing and data interpretation

7.6 C.t values

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REFERENCES

Figures and Tables

Table 7.1: UV dose requirements for virus inactivation credit

Table 7.2: C.t values for inactivation of viruses by free chlorine, pH 6 – 9

Table 7.3: C.t values for inactivation of viruses by chloramine

Table 7.4: C.t values for inactivation of viruses by chlorine dioxide, pH 6 – 9

Table 7.5: C.t values for inactivation of viruses by ozone

 

7.1 INTRODUCTION

No maximum acceptable values (MAVs) have been set for human viruses in the Drinking-water Standards for New Zealand (2005). It is likely that a MAV or MAVs will be established in a future edition. This chapter foreshadows such developments.

In the absence of any MAVs for viruses in the current DWSNZ it should be understood that if they are specifically sought, they should not be detected. If detected, advice should be sought from the relevant health authorities.

There are more than 140 different types of human enteric viruses that may contaminate potable source waters. These include several important groups: Hepatitis A virus, Hepatitis E virus, norovirus, enterovirus and adenovirus, that have been associated with waterborne illness and are capable of causing severe, and in some cases fatal, infections.

Human viruses are obligate intracellular parasites, which means they cannot grow or multiply outside their host. Viruses consist of a nucleic acid genome surrounded by a protein capsid and, in some cases, a lipoprotein envelope. These viruses are very small, ranging from 20 – 70 nm in diameter.

Human enteric viruses are shed in the gut, respiratory tract and occasionally urine of an infected person, and are discharged with body wastes into wastewater. Infected people do not always show signs of illness (asymptomatic) but they will still produce virus in their wastes. Specific viruses or strains of viruses are not always present in a community at any one time, but representatives of the large groups (e.g. adenovirus or enterovirus) are generally present on most occasions.

Enteric viruses may be found in high numbers in domestic wastewater. Recent New Zealand studies have shown adenovirus and enteroviruses to be present in concentrations greater 10,000 infectious virus units per litre of wastewater (Watercare Services Surrogate Study 2002). The numbers of viruses in wastewater varies with the level of virus infection in the community but, in general, human viruses will always be present in wastewater, even of small communities, (average human virus load 100 – 1000 infectious viruses/L) and will occasionally reach very high levels (>10,000 infectious virus/L) (Lewis et al 1986). Wastewater treatment processes that do not include a disinfection step are often inefficient in removing viruses (<90% removal) and some viruses may reach potable water supplies.

Human enteric viruses cannot multiply in the environment once outside the host. The viruses are characterised by the ability to survive for days, weeks or more, in the environment depending on the type of water, season and other factors (Hunter 1997).

A large proportion of the human viruses present in source drinking-waters will normally be removed or inactivated by well-operated standard drinking-water treatment processes.

Routine monitoring for viruses in treated water and source water is currently impractical in most situations in New Zealand because of the high cost of sampling and analysis, and problems of detection of a full range of the viruses occurring.

 

7.2 Health significance of human viruses in drinking-water

Hepatitis A virus, Hepatitis E virus, norovirus, enterovirus and adenovirus may occur in drinking-water where they are present in the source water and when water treatment does not remove them completely. Very few human enteric viruses (1 – 50 virus particles depending on type) are required to produce an infection in a susceptible person (Hunter 1997). The symptoms generally attributed to enteric viruses are gastroenteritis and diarrhoea but they can also cause respiratory, central nervous system, liver, muscular and heart infections. Some waterborne viruses have also been associated with some forms of diabetes, chronic fatigue syndrome and dementia (Nwachcuku and Gerba 2004). The major groups of viruses contaminating water are discussed below but may not represent all the viruses likely to be transmitted by water. It is reasonable to expect that further important groups of waterborne viruses will be detected in the future and that these will most likely cause atypical waterborne disease (Nwachcuku and Gerba 2004).

Norovirus: this group of caliciviruses includes the Norwalk and Norwalk-like viruses. Members of this group are strongly associated with waterborne outbreaks in many parts of the world. Symptoms of infection are mild and self-limiting and include vomiting, diarrhoea and nausea over 24 – 48 hours. Norovirus is quite prevalent in New Zealand and is responsible for a large proportion of viral gastroenteritis reported to health authorities (ESR 2004). This virus is one of the easiest to link to a common source outbreak as the symptoms occur rapidly after contact with the virus (approximately 24 hours).

Hepatitis A and E: Hepatitis A and E have a relatively low occurrence in New Zealand (ESR 2004) but induce quite significant symptoms including fever, malaise anorexia and jaundice. The disease is generally self-limiting but has a 2 percent mortality rate. The infectious doses for these viruses are relatively low (10 – 100 viruses) and symptoms do not occur until 10 – 50 days after infection. Internationally Hepatitis A and E outbreaks have frequently been associated with water.

Enteroviruses and adenoviruses: these two different groups represent the viruses that are most commonly found in contaminated surface water. These viruses produce a very broad range of symptoms including respiratory, skin and eye, nervous system, liver, heart and muscular involvement. Gastroenteritis with vomiting and diarrhoea is a less common outcome of infection with these viruses and is limited to only a few adenovirus and enterovirus types. Reported waterborne outbreaks of these viruses, other than in swimming pools, are very infrequent. It is not clear whether lack of reporting is because the dominant symptoms produced by these viruses are not those traditionally associated with water or food borne disease, or because such outbreaks are indeed rare (Hunter 1997).

Virus infections resulting from treated water have not been reported in New Zealand (ESR 2004) in recent years although internationally such outbreaks are recognised (Hunter 1997). Human viruses have been reported to occur at very low levels (0.1 – 1/100 L) in conventionally treated drinking-water in many countries (Vivier et al 2004) including New Zealand (Kim 2005).

Estimations of viral disease risk using standard risk assessment techniques with a high infectivity virus predict the surprisingly high annual risk of infection of between 1:3 and 1:25 from conventionally treated drinking-water contaminated by viruses at low levels (~1 virus per 100 litres) (Gerba and Rose 1992).

 

7.3 Occurrence of Human viruses in Source waters

The New Zealand freshwater microbiology study (McBride et al 2002) is the most significant study of human viruses occurrence in surface water in New Zealand to date. This study carried out in collaboration between the Ministries for the Environment, Agriculture and Forestry, and Health tested recreational water locations on 25 rivers and lakes every 2 weeks for 15 months.

Human adenovirus and/or enterovirus were detected, by qualitative molecular methods, in more than 50% of the 275 samples collected. This data suggests that human virus occurs quite frequently in surface waters and in a wide range of source water locations and types.

Subsequent culture based studies of virus occurrence in the Waikato River show that adenovirus and enterovirus levels are generally low, less than 5 per 100 L, but on some occasions may be as high as 10 per 100 L (Watercare Waikato River Monitoring studies 2003 - 2004).

Studies using sensitive qualitative molecular-based virus detection methods suggest that adenovirus occurrence may be 10 times higher than this level on some occasions in the Waikato River (Kim et al 2005) although it is not clear whether all of these viruses are able to produce infections.

International data collated by WHO suggest that typical surface source waters may contain 0 - 10 viruses per litre (WHO 2004).

 

7.4 Risk management

Potential for disease outbreaks associated with human virus contamination of source waters, and the potential for carry over to treated drinking-water is recognised throughout the developed world. Approaches to controlling the risks are largely through protection of source water quality by control of human activity in reservoir catchments, and through adequate treatment and disinfection of drinking-water. It is now well accepted that bacterial indicators such as E. coli are not adequate surrogates of viral occurrence. Human viruses tend to be more resistant to environmental stresses and water treatment mechanisms than are bacterial indicators, so the absence of the indicator may not equate with absence of the virus contaminant.

7.4.1 International approaches

The paucity of knowledge on the specific occurrence of human viruses in source waters, and the problems of virus detection and regular monitoring, mean that most guideline documents include only the qualitative requirement that, if tested for, human viruses should not be detected in treated drinking-water.

Where virus guidelines or standard requirements are in place these are stated either in terms of virus occurrence, or as water treatment plant virus removal efficiency. Such values are either derived from acceptable levels of health risk or, pragmatically, reflect virus detection capability.

Recent standard and guideline recommendations have moved towards risk-based evaluation of water treatment requirements. The USEPA Surface Water Treatment Rule includes a virus treatment requirement and requires that treatment of both filtered and unfiltered water sources is sufficient to remove or inactivate 99.99% (4 log) of viruses (USEPA 1994). This requirement is principally based on the acceptable (USEPA 1994) level of waterborne illness in a community (1 case per 10,000 consumers) and the likely level of viruses in surface water. Recent US proposals for surface water disinfection (USEPA 2003a) use the adenovirus group as the target virus.

The WHO Guidelines recognise that water treatment requirements will differ for different communities, and propose a risk-based approach for setting performance targets for surface water treatment plants (WHO 2004).

The risk-based approach takes into account a broad range of factors including virus occurrence and infectivity, water type, community health status and treatment characteristics. Such an approach requires a detailed knowledge of the water supply, water treatment performance and community activities and health status.

Approaches to managing viruses in treated water also recognise that the greatest health risk to a community occurs when water treatment conditions are atypical such as when source water condition is unusual, very high levels of virus occur, or through poor performance, or even failure, within the water treatment process.

7.4.2 Virus removal by current Water treatment processes

Reduction of virus numbers in water as a result of treatment can occur through either virus removal or virus inactivation. Each virus type may react somewhat differently to particular water treatment methods, but the bulk of research to-date suggests that some broad generalisations can be made.

Virus removal can occur by physical association of a virus with other particles. Particle flocs containing viruses are then removed by settlement or filtration. Virus association with particles and flocs may be enhanced by addition of coagulants and certain salts. The extremely small size of viruses means that they are unlikely to be removed if they are not associated with other particles. Water treatment processes such as flocculation, sand filtration, microfiltration and ultrafiltration, and prolonged standing in reservoirs, will result in physical removal of particle-associated viruses. Only reverse osmosis and dialysis membranes have pore sizes small enough to trap virus particles that are not associated with larger particles or flocs.

The effectiveness of virus removal is affected by those factors that act against particle association or floc formation including water condition and pH (LeChevelier and Au 2004).

Virus inactivation occurs through disruption of the external protein coat (capsid), modification of specific surface sites needed for infection (host receptor recognition sites) or major change to the nucleic acid (RNA or DNA). Disinfectants such as chlorine, chlorine dioxide, and ozone will cause disruption of the virus coat and of the exposed nucleic acids (Shin and Sobsey 2003, Tree et al 2003). Ultraviolet light in the range of 200 – 310 nm (antimicrobial range) will disrupt the nucleic acids by causing cross-linking that leaves them unable to replicate.

Viruses can also be inactivated by prolonged holding in reservoirs exposed to sunlight, elevated temperature and extremes of pH (e.g. lime treatment) (Sobsey 1989). Different virus types and strains will show different levels of resistance to chemical or physical inactivation. Adenoviruses are considered to be the most resistant virus group to many disinfection treatments, because of its structure and nucleic acid makeup, and have been used by the USEPA as a model virus for designing UV criteria for surface water treatment (USEPA 2003a).

The potential for virus inactivation by disinfectants is reduced by the presence of other particles or organic matter that will consume disinfectants or of light adsorbing or blocking materials that reduce UV penetration (LeChevelier and Au 2004).

Repair of disinfection damage is unlikely to occur in viruses as they do not have repair mechanisms as such. It has been suggested that some viruses (e.g. adenovirus) may be able to repair their DNA if there is no damage to the virus coat and they are able to infect a human cell (Nwachcuku and Gerba 2004).

Water treatment plants will normally include both virus removal and virus inactivation processes that act as multiple barriers.

Virus removal and inactivation efficiencies for a range of water treatment processes are reviewed in the WHO (2004) Guidelines (chapter 7), and by LeChevallier and Au (2004).

 

7.5 Sampling, testing and data Interpretation

The determination of virus removal efficiency within a water treatment plant, or occurrence in treated water, is dependant on the ability to reliably detect and enumerate the viruses. Determination of the health risk that viruses pose to the community using the water further depends on the ability to demonstrate or infer that the viruses detected are capable of causing human infection.

Virus detection and enumeration. No single method allows detection of all virus types and strains. Traditionally viruses have been concentrated from water samples using filtration or adsorption based techniques with subsequent detection by culture in a permissive human or primate cell line. Many of the virus concentration techniques were developed using poliovirus or other enterovirus types and it is not clear how effectively these work for other virus types particularly norovirus.

Virus concentration from large volumes of water is laborious and time consuming and adds significantly to the cost of virus analysis. Not all virus types are culturable in cell lines, again norovirus has not been cultured routinely and is not detectable using traditional methods.

Viruses (culturable and non-culturable) can be detected at very low levels using polymerase chain reaction (PCR) based molecular methods that target novel DNA or RNA sequences in the genetic information of the virus. Virus assay using PCR can target individual viruses or groups of viruses and multiple analyses would be required to investigate all the relevant viruses in a particular sample (Greening et al 2002). Recent advances in real-time PCR have made these methods both rapid and quantitative and potentially quite routine. PCR based methods are around 10-fold less sensitive that culture based methods for virus detection (Lewis et al 2000). Quantitative PCR based molecular methods are also significantly less expensive than traditional culture methods. It is unusual for a virus concentration and detection method to consistently recover more than 50 - 60% of the virus present in a sample (Lee and Jeong 2004).

Virus sampling strategies. Relatively few viruses are needed for an infection to occur in a susceptible person so low numbers of viruses must be quantified in relatively large volumes of finished water. For example, if source waters contain 5000 viruses per 100 L it would be necessary to sample and analyse at very least 200 L of finished water to demonstrate a 4 log reduction in viruses. Typically, source water sample volumes should be 10 – 100 L, partially treated waters 50 – 200 L, and finished, disinfected water sample volumes 100 – 200 L.

The current cost of virus analysis may make regular monitoring beyond the means of many groups responsible for drinking-water treatment.

Specific short-term studies of virus occurrence and inactivation/removal within a plant are feasible but should be designed carefully to allow adequate interpretation of the data.

Determination of virus infectivity. Molecular methods for virus detection do not specifically show whether viruses are still infectious. Detection of viruses using a cell-culture based technique shows that the viruses are infectious and pose a risk of illness to water consumers. Infectivity of a virus can however be inferred for certain RNA viruses (norovirus, enteroviruses, Hepatitis A and E) from molecular detection data where the viruses are subjected to chemical disinfection, but not UV disinfection (Greening et al 2002). Virus viability is inferred whenever virus nucleic acid is detected because the nucleic acids (single stranded RNA) are extremely susceptible to degradation in the environmental.

Interpretation of virus detection and occurrence data. Where viruses are detected in finished drinking-water the response to the data should be based, in consultation with relevant health authorities, on a risk evaluation incorporating the type and number of virus detected, the reproducibility of the result, and the health status and vulnerability of the community.

 

7.6 C.t VALUES

Appendix C of the LT1ESWTR Disinfection Profiling and Benchmarking Technical Guidance Manual (USEPA 2003b) includes C.t tables for disinfection of viruses by various disinfectants. These tables are referenced to AWWA (1991) but in the text of USEPA (1991) it would appear that it was a USEPA publication. Because the 2003 publication still uses the 1991 tables it is assumed that the data the 1991 tables were based on is still the latest information!

The USEPA Surface Water Treatment Rule required (inter alia) that treatment of both filtered and unfiltered sources remove or inactivate 4 log (99.99%) of viruses. This requirement was enacted in 1989. Presumably the 1991 tables were developed to assist water suppliers assess the degree of disinfection of viruses.

USEPA’s LT2ESWTR (2003a) includes a table showing the C.t values for disinfecting viruses using UV light. The proposed UV doses for inactivation of viruses were based on the dose-response of adenovirus because, among viruses that have been studied, it appears to be the most UV resistant and is a widespread waterborne pathogen. Health effects of adenovirus are described in Embrey (1999).

It is doubtful that this same approach was used in developing the 1991 tables; viruses are simply referred to collectively, and viruses are not defined in the 1991 information provided. Some viruses require a much higher C.t than others. Nor is it explained whether the data relate to studies in single virions or cell-associated virions – the latter require a higher C.t.

Table 7.1 shows the UV doses that water suppliers must apply to receive credit for up to 4 log inactivation of viruses. This is Table IV – 21 in USEPA (2003a).

 

Table 7.1: UV dose requirements for virus inactivation credit

Log Credit

Virus1

UV Dose (mJ/cm2)

0.5

39

1.0 (90% removal)

58

1.5

79

2.0 (99% removal)

100

2.5

121

3.0 (99.9% removal)

143

3.5

163

4.0 (99.99% removal)

186

1 based on adenovirus studies

Tables 7.2, 7.3, 7.4, 7.5 have been taken from Appendix C of USEPA (2003b) and copied from the 1991 publication, i.e. they refer to undefined viruses.

Based on Table 7.2, a free available chlorine content of 0.20 mg/L after 30 minutes retention time is equivalent to a C.t of 6. This would achieve 4 log inactivations at 10°C. At 5°C the minimum retention time should be 40 minutes, or if that cannot be achieved, the residual free chlorine content should be increased to 0.30 mg/L.

 

Table 7.2: C.t values for inactivation of viruses by free chlorine, pH 6 – 9

Log inactivation

1°C

5°C

10°C

15°C

20°C

25°C

2

5.8

4.0

3.0

2.0

1.0

1.0

3

8.7

6.0

4.0

3.0

2.0

1.0

4

11.6

8.0

6.0

4.0

3.0

2.0

Table 7.3: C.t values for inactivation of viruses by chloramine

Log inactivation

1°C

5°C

10°C

15°C

20°C

25°C

2

1243

857

643

428

321

214

3

2063

1423

1067

712

534

356

4

2883

1988

1491

994

746

497

Table 7.4: C.t values for inactivation of viruses by chlorine dioxide, pH 6 – 9

Log inactivation

1°C

5°C

10°C

15°C

20°C

25°C

2

8.4

5.6

4.2

2.8

2.1

1.4

3

25.6

17.1

12.8

8.6

6.4

4.3

4

50.1

33.4

25.1

16.7

12.5

8.4

Table 7.5: C.t values for inactivation of viruses by ozone

Log inactivation

1°C

5°C

10°C

15°C

20°C

25°C

2

0.9

0.6

0.5

0.3

0.25

0.15

3

1.4

0.9

0.8

0.5

0.40

0.25

4

1.8

1.2

1.0

0.6

0.50

0.30

 

REFERENCES

Embrey, M. (1999). Adenovirus in drinking water, literature summary. Final report. Prepared by The George Washington University School of Public Health and Health Services, Department of Environmental and Occupational Health, Washington, DC.

ESR (2004). Annual Summary of Outbreaks in New Zealand: 2003. Report to Ministry of Health ISSN 1176-3485.

Gerba C. P. and J. Rose (1992). Estimating viral risk from drinking-water: in Comparative Environmental Risk Assessment. Ch. 9, pp 117 – 137. CR Conthern Lewis Publishers.

Greening G., J. Hewitt and G. Lewis (2002). Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples. Journal of Applied Microbiology, 93, pp 745 – 750.

Hunter, P. (1997). Viral gastroenteritis. in waterborne disease. Epidemiology and Ecology. Chapter 28, pp 222-231. John Wiley and Sons.

Kim J. (2005). Human adenovirus in the Waikato River: Implication for water supply and public health. MSc thesis. University of Auckland Library.

LeChevelier M., K-K. Au (2004). Water treatment and pathogen control: Process efficiency in achieving safe drinking-water. WHO Drinking-Water Quality Series. WHO, Geneva.

Lee, H. K. and Y.S. Jeong (2004). Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliablity of waterborne virus detection. Applied & Environmental Microbiology, 70, pp 3632-3636.

Lewis, G. D., F. J. Austin, M. W. Loutit and K. Sharples (1986). Enterovirus removal from sewage - the effectiveness of four different treatment plants. Water Research, 20, pp 1291 - 1297.

Lewis G., S. L. Molloy, G. E. Greening and J. Dawson (2000). Influence of environmental factors on virus detection by RT-PCR and cell culture. Journal of Applied Microbiology, 88, pp 633-640.

McBride, G., D. Till, T. Ryan, A. Ball, G. Lewis, S. Palmer and P. Weinstein (2002). Freshwater Microbiology Research Programme Report: Pathogen Occurrence and Human Health Risk Assessment Analysis. Ministry of Health, Wellington.

Ministry of Health (2005). Drinking-water Standards for New Zealand 2005. Ministry of Health, Wellington.

Nwachcuku N. and C. P. Gerba (2004). Emerging waterborne pathogens: can we kill them all? Current Opinion in Biotechnology, 15, pp 175-180.

Shin G-A and M. D. Sobsey (2003). Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by ozone disinfection of water. Appl. Environ. Microbiol., 69 (7), pp 3975-3978.

Sobsey M. D. (1989). Inactivation of health-related microorganisms in water by disinfection processes. Water Science and Technology, 21 (3), pp 179-195.

Tree J. A., M. R. Adams, D. N. Lees (2003). Chlorination of indicator bacteria and viruses in primary sewage effluent. Applied & Environmental Microbiology, 69 (4), pp 2038-43.

USEPA (1994). National Primary Drinking Water Regulations: Enhanced Surface Water Treatment Regulations. 59 FR 38832; July 29.

USEPA (2003a). Long Term 2 Enhanced Surface Water Treatment Rule; Proposed Rule. National Primary Drinking Water Regulations: 40 CFR Parts 141 and 142, August 11, 2003.

USEPA (2003b). LT1ESWTR Disinfection Profiling and Benchmarking Technical Guidance Manual. EPA 816-R-03-004, Office of Water, May 2003. Available at: http://www.epa.gov/safewater/mdbp/pdf/profile/lt1profiling.pdf

Vivier J. C., M. M. Ehlers and W. O. Grabow (2004). Detection of enteroviruses in treated drinking-water. Water Research, 38 (11), pp 2699-705, 2004.

Watercare Services Ltd (personal communication): Surrogate study: Mangere wastewater treatment plant 2002.

Watercare Services Ltd (personal communication): Adenovirus and enterovirus monitoning data, Waikato River at Mercer, 2003, 2004.

World Health Organisation (2004). Guidelines for drinking-water quality, 3rd edition. Volume 1: recommendations. ISBN 92 4 154638 7. World Health Organisation, Geneva.

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HUMAN IMMUNODEFICIENCY VIRUS: VIROLOGY AND VACCINE DEVELOPMENT

 

Elizabeth A. Cahill

Abstract. Figures from 2004 suggest that as many as 42.3 million people, 1.1 percent of the world’s population, are currently infected with the human immunodeficiency virus (HIV). In Sub-Saharan Africa, the region suffering most from this pandemic, it is estimated that one in four adults will be killed by acquired immune deficiency syndrome (AIDS) (deWaal, 2004). HIV is a Lentivirus that infects T-helper cells, macrophages and monocytes. The host immune system reacts by removing its own infected T-cells, making the patient more susceptible to opportunistic infection. Chemotherapeutic drugs may drastically reduce morbidity and mortality of patients. These are available to less than 2% of persons with advanced AIDS. Despite much research into various types of vaccines, an effective vaccine against HIV has yet to be developed.

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15 September 2004

Research sponsored by DARPA Grant DAAD19-02-1-0288, P00001

1 September 2004

Reed College, Portland, OR

DARPA Grant


Introduction

Just under twenty five years have passed since the Human Immunodeficiency Virus (HIV) was first described (Barre-Sinoussi et al., 1983) and already twenty million people have died (UNAIDS global report, 2004), approximately 14 times the population of the island of Manhattan (US Census data 2000). Despite the enormous amount of research that has gone into vaccine and therapy development, no cure has been found and the number of people with HIV continues to grow. At the close of the year 2003, UNAIDS estimated that 42.3 million people were currently infected. Of the twenty million people dead, approximately 2.9 million died last year alone (Steinbrook, 2004). This pandemic is changing the face of the world.

The enormity of these numbers makes them difficult to comprehend. Some more staggering numbers help to make this figure more accessible. Among adults aged 15-49, 1.1 percent are currently infected (Steinbrook, 2004). Each day approximately 14,000 new infections are established; 95% of new infections occur in developing countries (Emini and Koff, 2001). Every country in southern Africa reports HIV infection rates ranging from 20%-35% (deWaal, 2004). In sub-Saharan Africa approximately 1/3 of children (under age 15) have lost one or both of their parents. In some countries in Africa, there are more than one million orphans (Lewis, 2004). Reversing these trends will be an enormous struggle but is tremendously important. The implications of these numbers and the social challenges surrounding this epidemic cannot be discussed in this paper. However, it is important to consider the macroscopic reality of the HIV crisis when considering the virology of the disease. This virus has created a crisis that demands a global effort. For scientists, politicians and individual citizens it must simultaneously be treated as a pressing crisis and a long-term reality. Both types of strategies are essential.

 

Basic virology

Human Immunodeficiency Virus (HIV) is an infection of the immune system. Other immune system infections include Lupus, asthma and Crohn’s disease. To date, no human has been able to overcome an infection with HIV, although some persons have been able to force viral loads to below the level of detection. The most common modes of HIV infection are direct blood exchange (intravenous drug use or blood transfusion), sexual contact and mother to child transmission. Each of these infection routes can be dampened with appropriate protection strategies.

An understanding of the basic virology of HIV is necessary for discussions of vaccine and chemotherapeutic developments and challenges. HIV is a retrovirus. This indicates that it is an enveloped RNA virus that uses the enzyme reverse transcriptase (RT) to convert its viral RNA into a complementary DNA (cDNA). The resultant cDNA, during a successful infection, is inserted into the host chromosomal DNA where it is able to utilize host machinery, and energy, to further replication and infection. One important feature of RT is that it is error-prone. This serves to increase the virus’ genetic variability and the rate of variant evolution. Retroviral infection can cause numerous diseases, malignancies and cancer. HIV belongs to the genus Lentivirus (“slow virus”), a subset of the family Retroviridae. Lentiviruses are generally larger than other retroviruses and, as their name suggests, have long incubation periods. Every lentivirus causes immune deficiencies and nervous system dysfunctions (Flint et al., 2000) and can be responsible for malignancies such as arthritis or autoimmune disorders.

For an infection to be established the virus must adsorb to the host cell surface. Therefore, the availability of surface receptors determines the host and tissue specificity of any viral infection. In HIV the major viral receptor is a cell surface CD4 protein found on T-helper cells, macrophages and monocytes (Prescott et al., 2001). The humoral and cellular host immune systems both respond to the new infection. The humoral immune system is responsible for the production of antibodies again HIV-1. Antibodies bind to the virus, targeting them for destruction. The cellular immune response is activation of cytotoxic T lymphocytes (CTLs) that directly remove cells presenting viral antigens. The combined efforts of these two limbs of the immune system are insufficient to clear the virus. The continued viral replication in the cells of the lymph nodes ultimately leads to the destruction of the host lymph node structure (Prescott et al., 2001).

The fact that the virus targets both regulatory and antigen presenting immune system cells partially explains the virus’ total ability to avoid destruction by the host immune system (Emini and Koff, 2004). A coreceptor is necessary for viral fusion to the host membrane; it is the binding ability of the coreceptor that determines the tropism of the HIV strain (Flint et al., 2000). During the initial stages of HIV infection the corecptor to the moncyte or macrophage CD4 (M-tropic), CCR5, is essential. Persons homozygous for a mutated, and therefore nonfunctional, CCR5 receptor are impervious to HIV infection (Sullivan et al., 2001). Those heterozygous for the CCR5 receptor maintain a lower viral load during the pre-AIDS course of the disease and appear to progress to AIDS more slowly (Prescott et al., 2001). Mutations in the CCR5 receptor are extremely rare in many regions, such as sub-Saharan Africa and Asia, a fact that may alter the face of HIV/AIDS epidemiology in those regions (Sullivan et al., 2001). After the virus has been established in the host system, the T-cell tropic fusin (CXCR-4) protein is the coreceptor that determines the success of the virus (Prescott et al., 2001). Fusion facilitates the formation of syncitia. This is not seen in M-tropic strains.

All retroviruses have three common structural proteins Gag, Pol, and Env, which code for the core proteins and structural components of the virion, the reverse transcriptase and the envelope glycoprotein, respectively (Flint et al., 2004). Once the HIV-1 virus gains access to the host system, the viral gp120 Env protein binds the CD4 plasma membrane receptor on a host T lymphocyte. The gp120 protein is inaccessible to the host antibodies when it is unattached. It is able to bind CD4 only after a structural adjustment that exposes part of the gp120 to a chemokine receptor, such as CCR5 (Emini and Koff, 2004). The exposed part of the gp120 remains protected from antibodies by either steric hinderance or extensive glycosylation (Emini and Koff, 2004). Every fusion event is followed by the release of the virion core and RNA strands into the cytoplasm of the host T lymphocyte cell. The viral RNA is translated by viral RT into a single-stranded DNA (ssDNA). The RNA strand is then degraded; the ssDNA is used as a template to create a double-stranded DNA (dsDNA). At this point the virus either becomes latent in the cell or forces the cell to transcribe viral mRNAs. The formation of new virions within a host cell will ultimately destroy it, facilitating the release of thousands of new virions into the host system.

Because infected T cells are removed by the host’s own immune response, the patient’s immune response is necessarily compromised. As the loss of T cells becomes more advanced, the infected person becomes increasingly vulnerable to opportunistic infections because their immune system is less functional. These opportunistic infections are often the cause of patient mortality. The progression of HIV infection may be more aggressive in developing countries such as those in sub-Saharan Africa because of chronic infections with pathogens and parasites. Growing antibiotic resistance compounds this problem, making the treatment of other infections more challenging. However, the success of CTLs in removing the infected cells is not wholly destructive. The ability of the patient to clear infected cells, and therefore set the level of viremia during the asymptomatic phase, is a determinant of long-term HIV control. Therefore, the CTLs exert a strong selective pressure, which drives the formation of viral escape mutations (Leslie et al., 2004). HIV is also believed to carry oncogenes; cancer is another common demise of HIV patients. The central nervous system (CNS) can also be damaged because the virus is small enough to cross the blood-brain barrier.

There are roughly four stages described for the average course of an HIV infection. During the primary stage of infection the body produces an acute response to the introduction of the virus. Clinically, this period is best described by a general malaise but HIV antibodies can be detected in the body at this point and the patient is now infectious for the remainder of their lives. Normally, the host system manages to gain control over viral replication at this point and the viremia falls. The virus is not cleared but evades the host immune response and establishes a persistent infection (Emini and Koff, 2004). The patient is often asymptomatic during this phase, which lasts for a variable amount of time. Only approximately 10% of HIV positive adults exhibit disease progression in the first two or three years of infection. After ten years, 80% of HIV positive adults have signs of disease progression (Ho, 1997). During this period of clinical latency, destruction of the lymphoid tissues continues as the virus replicates (Fauci, 2003). The length of the asymptomatic phase poses an enormous challenge for disease control and epidemic modeling (Anderson and May, 1990). Eventually, the virus does overcome the host immune system and the symptoms of HIV/AIDS begin to emerge, the number of host T-cells drops and the patient historically will progress to AIDS at this point (T-cell count below 200 units). No survivors of AIDS have yet been recorded. The symptoms of AIDS are variable but can often include: fever, weight loss, skin rashes, diarrhea, dementia, myelopathy, peripheral neuropathy and increased susceptibility to any opportunistic pathogen (Fauci, 2003). More rare symptoms include Kaposi sarcoma, oral hairy leukoplakia and lymphomas (Flint et al., 2004).

Chemotherapeutic drugs have changed the quality and quantity of life that can be expected for HIV-positive persons who can afford to take them. The three general types of palliative drugs are RT inhibitors that are nucleoside analogs, nonnucleoside inhibitors of RT, fusion inhibitors and protease inhibitors (Prescott et al., 2002). The first effective treatment used to combat HIV was azidovudine (AZT), which is a RT inhibitor (McCleod and Hammer, 1992). A combination of these three types of drugs is most effective and results in greatly reduced morbidity and mortality in countries where they are available (Fauci, 2003). However, the virus does remain latent in T-helper cells and treatment must be continued indefinitely to avoid activation of the virus.

 

Vaccine development

In addition to the continued arms race for improved chemotherapeutic agents, a vaccine will be a necessary step towards slowing down the spread of this virus. Despite great efforts an effective vaccine remains elusive. Vaccines have historically been most effective in blocking diseases that have a period of acute illness such as smallpox, polio or tetanus. The fact that the host immune response has never been effective in clearing this virus indicates the challenge that biologists face in vaccine development. However, with approximately 14,000 new infections each day (Emini and Koff, 2004) it is a task of enormous importance.

An effective vaccine will have to induce, upon introduction of the virus, an immune response that is different, not just stronger or faster, than the naïve immune response. An obvious target in vaccine development is the Env surface protein, which is integral in establishing HIV infection, as discussed above. A vaccine based on the humoral arm of the immune system would produce antibodies to block new infections. A trimeric Env complex, which includes gp120 and gp41, triggers fusion when complexed with the CD4 receptor and an appropriate coreceptor. Neutralizing monoclonal antibodies (mAbs) have been developed for gp120 that interfere with binding to the CD4 receptor. Similarly mAbs designed for gp41 seem to block fusion. Neutralizing antibodies have been developed for many distinct regions in these glycoproteins but have not been effective in vivo (Burton et al., 2004). Gp120 quickly adapts to immune pressure forming variable surface proteins and gp41, although considered to be more stable, is kinetically inaccessible (Burton et al., 2004). Fortunately, there is a simian immunodeficiency virus (SIV) that is almost identical to HIV. Unfortunately, an effective vaccine has not yet been developed to withstand an SIV challenge (Desrosiers, R.C. 2004).

Another notable obstacle in the vaccine development effort is the sequence variability of the HIV-1 viruses to which humans are exposed. Goulder and Walker (2002) described cases where patients already infected with one strain of HIV-1 became infected again when exposed to a different strain. This has serious consequences for treatment as well as vaccination. In cases of superinfection, the same drug regime that is effective for one strain may not be effective for the other. The antiretroviral drugs used for treating HIV have severe side effects for many patients and increasing the drug dosage or number of drugs may not be possible. A fully effective vaccine would have to protect against many different strains of HIV.

A vaccine designed for a cellular immune response would create anti-HIV-1 CD4+ memory and CD8+ CTLs (Emini and Koff, 2004). The humoral response would, theoretically, be more important to block the establishment of an infection. However, a stronger, faster cellular immune response could help to establish a lower viral load. This is important both for the morbidity of individuals and epidemiologically (Emini and Koff, 2004). This should not be undervalued. The fact that teenagers in every country in sub-Saharan Africa have a 50% chance of being infected with HIV (deWaal, 2002) makes it important to use realistic triage tactics in addition to seeking out a totally effective vaccine.

Microbicides are another strategy being pursued. An entirely effective microbicide (non-toxic, contraceptive or non-contraceptive, fully effective) is many years away (Lewis, 2003). Yet even a partially effective microbicide could save millions of lives. Vaccine and microbicide development can be seen as an important strategy for protecting women (Lewis, 2003). In many cultures women have neither the ability to protect themselves from exposure to this virus nor the power to handle the consequences of the illness. Both a vaccine and a microbicide could give a woman the ability to protect herself without the necessary consent of her partner.

 

Conclusions

This epidemic is unlike any the human race has encountered before and must be handled as such. In 1981, during the conservative years under President Reagan, when doctors in New York and California began to note this remarkable and horrible illness in young, gay men, the reaction was slow (Shilts, R., 1987). Public fear and indifference to the gay community made the work of the doctors dedicated to uncovering the root of this disease a battle (Shilts, R., 1987). Similarly, in September 2001, President Mbeki of South Africa questioned the link between HIV and AIDS and decided that zidovudine was too toxic to distribute in clinics. This ignored substantial research demonstrating that zidovudine could dramatically reduce the mother to child transmission of HIV (Lallemant et al., 2004). This trend of insistent ignorance and cruelty is seen repeatedly. In Russia, a group of medical school graduates wrote, in 1997, "We are . . . categorically opposed to combating the `new disease' AIDS! We intend . . . to impede the search . . . to combat this `noble' epidemic. We are certain that . . . AIDS will destroy all drug addicts, homosexuals, and prostitutes. . . . Long live AIDS!" (Field, 2004). Despite all of this, there is great cause, and need, for optimism. The pandemic has reached such staggering levels that honest discussions and massive changes are beginning. Education programs, condom and needle distribution programs and health care programs are growing. The World Health Organization aims to have 3 million people in treatment by the year 2005. This is a step towards the ultimate goal of total access to antiretroviral medications. HIV introduces a nontraditional security threat in addition to the massive human tragedy. Countries with high incidence of HIV infection are experiencing severe economic crises along with enormous demographic changes. The effects of the epidemic in regions such as sub-Saharan Africa will be felt globally. There is a constant call for the change in the behavior of those in high-risk populations. For health considerations, this is valid. However, an equally substantial change in behavior is needed in the general population to make the necessary funding and support available.

 

References

Barre-Sinoussi, F., Chermann,. J.-C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. Isolation of a T-lymphotropic retrovirus from patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868-871.

Boes, M, Ploegh, H.L. Translating cell biology in vitro to immunity in vivo. Nature 430, 264-271 (2004).

Bretscher, M.T., Althaus, C.L., Muller, V., Bonhoeffer, S. Recombination in HIV and the evolution of drug resistance: for better or for worse? BioEssays 26.2, 180-188 (2004).

Coovadia, H. Antiretrovial agents- how best to protect infants from HIV and save their mothers from AIDS. NEJM, 351, 289-292 (2004).

deWaal, A. Fucking Soldiers: Militarisation, Secrecy and the AIDS pandemic in Africa. Justice Africa papers (2002).

Dezso, Z. and Barabasi A-L. Halting viruses in scale-free networks. Physical Review 65, 055103-1-4 (2002).

Fauci, A. HIV and AIDS: 20 years of science. Nature 9, 839-842 (2003).

Fawzi, W.W. et al. A randomized trial of multivitamin supplements and HIV disease progression and mortality. NEJM, 351, 23-32 (2004).

Field, M.G., HIV and AIDS in the former Soviet bloc. NEJM, 351,117-120 (2004).

Friedrich, T.C. et al. Reversion of CTL escape-variant immunodeficiency viruses in vivo. Nature Medicine 10, 275-281 (2004).

Goulder, P.J., Walker, B.D. HIV-1 superinfection – a word of caution. NEJM, 347, 756-758 (2002).

Lallemant, M. et al. Single-dose perinatal Nevirapine plus standard Zidovudine to prevent mother-to-child transmission of HIV-1 in Thailand. NEJM, 351, 217-227 (2004).

Leslie, A.J. et al. HIV evolution: CTL escape mutation and reversion after transmission. Nature Medicine 10, 282-289 (2004).

Merrell, D.S. and Falkow, S. Frontal and stealth attack strategies in microbial pathogenesis. Nature 430, 250-256 (2004).

McCleod, G.X., Hammer, S.M. Zidovudine: Five years later. Ann. Int. Med. 117, 487-501 (1992).

Oltvai, Z.N., Barabasi, A-L. Life’s complexity pyramid. Science 298, 763- 764 (2002).

Sarafianos, S.G., Hughes, S.H., Arnold, E. Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs. IJBCB 36, 1706-1715 (2004).

Shilts, R. And the Band Played On. Penguin, 1988.

Steinbrook, R. The AIDS epidemic in 2004. NEJM, 351, 115-117 (2004).

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Retinoic acid-mediated antibody response

Supplementary Information

Materials. Amino acid building blocks, resins and coupling agents were obtained from Novabiochem (Darmstadt, Germany), Anaspec (San Jose, CA) or ChemPep (Miami, FL). Cholesterol, dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Distearoylglycerol (DSG; LP-R4-029) was obtained from Genzyme Pharmaceuticals (Cambridge, MA). Anhydrous solvents of 99.8% or greater purity were obtained from Acros Organics (Geel, Belgium). Monophosphoryl lipid A derived from Escherichia coli (MPL; #L6638), all-trans retinoic acid (ATRA; #R2625) and 13-cis retinoic acid (13-cis RA; #R3255) were obtained from Sigma-Aldrich. Unless otherwise specified, all other reagents were obtained from Sigma-Aldrich.

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Lipopeptide synthesis. N-MPR peptide (NEQELLELDKWASLNGGK) was synthesized on NovaPEG resin in an automated solid phase synthesizer (ABI 433A, Applied Biosystems, Foster City, CA) with standard fluorenylmethyloxycarbonyl/o-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate/n-hydroxybenzotriazole (FMOC/HBTU/HOBT) protocols. An orthogonally protected lysine (Fmoc-Lys(1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl)-OH; Fmoc-Lys(ivDde)-OH) was incorporated at the C terminus for on-resin conjugation of lipids or biotin. The N terminus was Boc-protected. Removal of the ivDde group was accomplished by 3 x 15 minute treatments of the peptidyl resin with 2% hydrazine hydrate in dimethylformamide (DMF; 10 mL per g resin). The resin was washed in DMF (3 x 10 mL) and dichloromethane (DCM; 3 x 10 mL) and dried under vacuum.

Lipid conjugation was accomplished via amidation of a carboxylated lipid and a deprotected lysine e-amine at the C terminus. A carboxyl group was introduced to DSG via reaction of an available alcohol with succinic anhydride. Briefly, 1.8 mmol DSG was dissolved in 5 mL anhydrous DCM and combined with 3.6 mmol succinic anhydride in 10 mL anhydrous pyridine. The mixture was refluxed at 60 oC overnight. The reaction was continued to completion as monitored by thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS; Voyager DE, Applied Biosystems, Foster City, CA) in para-nitroaniline matrix. The product was washed twice with 1M hydrochloric acid (HCl), dried over sodium sulfate and stored dry until use. Carboxylated lipids were obtained in approximately 90-100% yield. The molecular weight and TLC RF value were as follows: DSG-Suc, 749.6 (+ Mg2+) Da, RF 0.08 (DCM/acetone, 20/1).

Lipidation was accomplished by activation of 270 mmol carboxylated lipid with 270 mmol each of HBTU, HOBT and diisopropylethylamine (DIEA) in anhydrous DMF/DCM (DCM as needed for lipid solubilization) for 30 min at room temperature followed by addition of 67.5 mmol resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with DMF (4 x 10 mL) and DCM (4 x 10 mL) to remove unreacted lipids and dried under vacuum. The lipopeptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% water, 2.5% ethanedithiol and 1% triisopropylsilane for 4 hours under argon. The lipopeptide was precipitated into cold ethyl ether, pelleted by centrifugation at 3000 rpm (RT6000, Sorvall, Waltham, MA) and washed once with cold ethyl ether. The ether was poured off and the pellet was re-dissolved in methanol (MeOH), transferred to a round bottom flask, dried by rotary evaporation under reduced pressure and further dried under high vacuum. Stock lipopeptide solutions were prepared MeOH/CHCl3 and stored at -20 oC. The molecular weight was as follows: N-MPR-DSG 2963.2 Da.

Biotinylated N-MPR peptide was prepared for use in ELISA by an analogous method. Biotin was attached to the deprotected C terminal amine by activation of 500 mmol D-biotin with 500 mmol HBTU/HOBT/DIEA in 1.65 mL anhydrous 1:1 DMF/dimethylsulfoxide (DMSO) for 30 min followed by addition of resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with 1:1 DMF/DMSO (3 x 10 mL), DMF (3 x 10 mL) and DCM (3 x 10 mL) and dried under vacuum. Biotinylated peptides were cleaved and purified as described above. Molecular weight of N-MPR-biotion was 2455.1 Da as determined by MALDI-MS. Biotin content was quantified by 4´-hydroxyazobenzene-2-carboxylic acid dye exclusion (Sigma #H2153) according to the manufacturer’s instructions.

 

RAL Synthesis. The synthesis of all-trans retinoic acid phospholipid (RAL) is depicted in Scheme S1. First, 1-O-octadecyl-2-O-benzyl-sn-glycerol was converted to phosphocholine by phosphorylation with phosphorus oxychloride and coupling to the choline tetraphenyl borate salt. Then the benzyl group was removed by catalytic transfer hydrogenation. Finally, all-trans retinoic acid was attached to the 2-hydroxy group with the typical DCC/DMAP method.

1-O-octadecyl-2-O-benzyl-sn-glycerol was from BACHEM (Torrance, CA). Other reagents were from Aldrich (Milwaukee, WI). TLC analyses were performed on 0.25-mm silica gel F254 plates using a variety of developing systems. High performance flash chromatography (HPFC) was carried out on a Biotage (Charlottesville, VA) Horizon™ HPFC™ system with pre-packed silica gel columns (60 Ǻ, 40-63 mm). Unless noted otherwise, the ratios describing the composition of solvent mixtures represent relative volumes. 1H NMR spectra were acquired on a Varian 400 MHz instrument. Chemical shifts are expressed as parts per million using tetramethylsilane as internal standard. J values are in Hertz. MALDI-TOF mass spectra were obtained at the Mass Spectrometry Facility, University of California San Francisco.

 

1-O-octadecyl-2-O-benzyl-sn-glycero-phosphate (1): A solution of 1-O-octadecyl-2-O-benzyl-sn-glycerol (10 g, 23 mmol) and anhydrous pyridine (3.72 mL, 2 equiv.) in anhydrous tetrahydrofuran (40 mL) was added dropwise to the freshly distilled phosphorus oxychloride (2.36 mL, 1.1 equiv.) in tetrahydrofuran (20 mL) with stirring at 0 °C. Stirring was continued for 3 h at 0 °C. Then 10% sodium bicarbonate (60 mL) was added, and the mixture was stirred at 0 °C for 15 min. The solution was then poured on ice water (200 mL), acidified with conc. HCl (pH ca. 2), and extracted with diethyl ether (400 mL × 2). The ether extracts were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, azeotropically dried with toluene twice, and used directly for next step reaction. Yield: 11.8 g, 100%. TLC: Rf = 0.05 (CHCl3/MeOH/NH4OH, 65/25/4).

 

1-O-octadecyl-2-O-benzyl-sn-glycero-phosphocholine (2): Compound 1(5.14 g, 10 mmol), choline tetraphenyl borate (8.46 g, 2 equiv.) and 2,4,6-triisoproylbenzene sulfonyl chloride (6.04 g, 2 equiv.) were dissolved in anhydrous pyridine (100 mL) with heating. The reaction mixture was stirred at 70 °C for 1h, then 3 h at room temperature. After the addition of water (10 mL), the solvents are removed by rotary evaporation. The residue was extracted with diethyl ether (250 mL × 2). The extracts were combined and evaporated. The crude product was purified by HPFC using a solvent gradient of 20% -35% MeOH-H2O (25/4) in chloroform. Yield: 5.8 g, 96.7%. TLC: Rf = 0.17 (CHCl3/MeOH/H2O, 65/25/4). 1H NMR (CDCl3-CD3OD), d 0.87 (t, J = 7.2, 3H); 1.27 (br, 30H); 1.57 (m, 2H); 3.15 (s, 9H); 3.36-3.60 (m, 7H); 3.83 (m, 1H); 4.04 (m, 1H); 4.16 (m, 2H); 4.68 (m, 2H); 7.31-7.45 (m, 5H). MALDI-MS calculated for C33H63NO6P+ [M + H]+ 600.45, found 600.54.

 

1-O-octadecyl-2-hydroxy-sn-glycero-phosphocholine (3): To as solution of compound 2(5.77 g, 9.6 mmol) in 70 mL methanol, were added 10% Palladium on activated carbon (2 g) and ammonium formate (3.03 g, 48 mmol) under nitrogen atmosphere. The mixture was stirred vigorously under nitrogen for 10 h at 60 °C. The mixture was cooled to room temperature, filtered through Celite 545. The filtrate was concentrated, and applied to a flash40+M column, purified with the HPFC system using an elution gradient of 30% -50% MeOH-H2O (25/4) in chloroform. Yield: 3.3 g, 67%. TLC: Rf = 0.05 (CHCl3/MeOH/H2O, 65/25/4). 1H NMR (CDCl3-TFA, 10:1), d 0.88 (t, J = 6.9, 3H); 1.27 (br, 30H); 1.63 (m, 2H); 3.25 (s, 9H); 3.64-3.85 (m, 6H); 4.30 (m, 1H); 4.58 (m, 1H); 4.68 (m, 2H); 5.03 (m, 1H). MALDI-MS calculated for C26H57NO6P+ [M + H]+ 510.40, found 510.62.

 

1-O-octadecyl-2-all-trans-retinoyl-sn-glycero-phosphocholine (4; RAL): To a solution of compound 3 (0.6 g, 1.1 mmol) and all-trans retinoic acid (0.73 g, 2 equiv.) in dry ethanol-free chloroform, were added dimethylaminopyridine (0.15 g) and dicyclohexylcarbodiimide (0.5 g, 2.1 equiv.). The reaction mixture was kept in dark with stirring for 36 h. The precipitate was filtered and the filtrate was concentrated, applied to flash 25+M column, purified with the HPFC using a elution gradient of 10%-30% MeOH/28% NH4OH (25/4) in chloroform. Yield: 626 mg, 72%. TLC: Rf = 0.52 (CHCl3/MeOH/NH4OH, 65/25/4). 1H NMR (CDCl3), d 0.89 (t, J = 6.9, 3H); 1.04 (s, 6H); 1.26 (br, 30H); 1.45-1.53 (m, 4H); 1.63 (m, 2H); 1.72 (s, 3H); 2.00 (s, 3H); 2.04 (m, 2H); 2.34 (s, 3H); 3.32 (s, 9H); 3.41 (m, 2H); 3.58 (m, 2H); 3.78 (m, 2H); 4.00 (m, 2H); 4.30 (m, 2H); 5.17 (m, 1H); 5.79 (m, 1H); 6.12-18 (m, 2H); 6.28 (m, 2H); 7.01 (m, 1H). MALDI-MS calculated for C46H83NO7P+ [M + H]+ 792.60, found 792.84.

 

RAL digestion by phospholipase A2. The cleavage of retinoic acid from RAL by phospholipase A2 (PLA2) was examined in vitro. 10 mmol RAL were dried to a thin film in a borosilicate glass tube by rotary evaporation under reduced pressure and following by further drying under high vacuum overnight. The film was hydrated in 2 mL containing 10 mM HEPES, 25 mM KCl, 10 mM CaCl2 and 20 mM Triton-X, pH 8.8 and heated at 45 oC for 20 min. Fifty mL of a 100 U/mL solution of snake venom PLA2 (Sigma #P7778) were added to 250 mL of the RAL solution and incubated for 30 min. When analyzed by TLC, incubation with PLA2 resulted in total loss of the original RAL spot (Rf = 0.38) and appearance of two new spots, corresponding to free retinoic acid (Rf = 0.28) and lysolipid (Rf = 0.18; CHCl3/MeOH/NH4OH, 65/25/4).

 

Liposome preparation. Lipopeptides were formulated in liposomes composed of 15:2:3 DMPC:DMPG:Cholesterol with MPL, RA and lipopeptide as indicated. Prior to use, glassware was rinsed with MeOH and CHCl3 and dried for at least 90 min at 150 oC to destroy pyrogens. Lipid solutions were combined in borosilicate glass tubes and dried to a thin film by rotary evaporation under reduced pressure. Films were further dried under high vacuum overnight. Lipids were hydrated in sterile PBS (UCSF Cell Culture Facility) by intermittent vortexing and bath sonication under argon for a brief period (approximately 15 seconds) to disperse the lipids into the buffer. Defined diameter vesicles were formed by extrusion 11 times through 400 nm polycarbonate membranes using a hand-held extruder (Avestin, Ottowa, Canada). To prevent contamination, the extruder was disassembled and thoroughly cleaned with MeOH and sterile PBS between samples. Vesicle size was characterized by dynamic light scattering and zeta potential was determined by electrophoretic mobility (Zetasizer 3000, Malvern, New Bedford, MA). Liposomes were prepared fresh prior to each injection and stored at 4 oC under argon until use.

 

Liposome association of N-MPR-DSG and RA. Liposomal association of antigen and retinoic acid was measured following sedimentation of liposomes by an ultracentrifugation method. Essentially, liposomes were formulated with antigen, MPL and RA exactly as described in Methods and centrifuged at 150,000g for 45 minutes in a Beckman TL-100 ultracentrifuge. When control liposomes containing fluorescently labeled lipids were centrifuged in this manner, greater than 90% of the fluorescence was consistently recovered in the liposome pellet. The liposome pellet was resuspended by pipetting and an aliquot was disrupted by mixing with 1.5% (v/v) C12E10 detergent. Antigen concentration in the pellet and supernatant was quantified by the absorbance of aromatic side chains at 280 nm and RA concentration was quantified by the absorbance at 350 nm (NanoDrop, Thermo Scientific, Wilmington, DE). The percent of liposome association was defined as (Pellet)/(Pellet + Supernatant) x 100.

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ASSIGNMENT SHEET FOR VIROLOGY TERM PAPER Spring 2010

Your assignment is to survey the primary literature in a well-focussed area of virology and to write a literature research paper (also known as a "term paper") of approximately 3000-4000 words reviewing the current state of knowledge in the chosen area. The purposes of the assignment are to acquaint you with the current literature in virology, provide you with in-depth knowledge in an area of virology that is interesting to you and to familiarize you with some "cutting edge" research in virology.

The term paper assignment is separate and distinct from the oral report assignment although there is some overlap in purpose between the two assignments.

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A: Selection of topic

There is no restriction as to topic except that it must involve some aspect of viruses and it must be approved by the instructor. The latter restriction is enforced to keep topics well-defined and manageable and to ensure that no two students select an identical topic.

The "first come first served" rule is in effect here. Students who make a topic selection early are less likely to run into conflicts with other students. Register your choice with the instructor to receive priority. Email is a good option here.

In order to keep to a schedule, the term paper topic must be elected fairly early in the semester. This means you will need to carry on some extra reading outside class and consult with the instructor as you define your interests and narrow your choices. The deadline date for topic selection is given in the Term Paper schedule.

Your term paper topic may be related to but not identical to the topic of your oral report. For graduate students, your term paper topic may be related to but not identical to your thesis topic. I will expect an OK from your major advisor that the term paper topic you have chosen is not too closely related to your thesis. These may be subsequently revised, if necessary.

 

B. Library Research

You will need to survey the primary literature in order to write your paper (and perhaps to select your term paper topic in the first place). Some scientific journals devoted to reporting the results of studies with viruses include (but are not limited to) the following.

Virology: publishes mechanistic and molecular reports on all types of viruses; perhaps the most prestigious virological journal

Journal of General Virology: publishes studies on all aspects of virology, including studies considered too biologically oriented to be accepted for Virology; because of the wide range of studies published here, this tends to be the most interesting virological journal

Journal of Virology: concentrates mainly on medical aspects of virology and on animal virology, but does publish studies on plant and bacterial viruses; papers with molecular, biochemical, structural, biological orientation may appear here

Intervirology: publishes fewer articles, but does accept a wide range of topics; this is the journal in which matters of taxonomy, nomenclature, classification are usually discussed

Phytopathology: publishes a few papers each issue pertaining to plant viruses, usually reporting biological studies

Journal of Bacteriology: publishes papers on various aspects of bacterial viruses

Journal of Clinical Microbiology: publishes a few articles each issue strictly on medical aspects of virology

Journal of Medical Virology: devoted to a wide range of aspects of medical virology, including case studies

Journal of Virological Methods: reports new methods for the study of animal, plant and bacterial viruses

Cell: the most widely-cited journal in all the biological sciences occasionally publishes an article pertaining to virology, when the studies reported have very great significance

These journals are by no means the only ones which contain papers on viruses. They are merely the leading journals in the field. Unfortunately, the IUP library receives few of them. Obtaining copies of articles which appear in other prominent journals is thus made somewhat problematic.

There are several ways to deal with this difficulty. The most effective way is to travel to a university-level library in another community (Pittsburgh, State College, Morgantown, to name a few) and look up the article there. The second way, which is less reliable and ultimately more time-consuming, is to use the inter-library loan service (ILLIAD). The third way, the slowest and least reliable of all, is to use reprint request cards to ask authors directly for a copy of the needed article. Your instructor will demonstrate the use of these cards. Another way that may prove effective is to download from the internet, when possible.

Your paper MUST be based mainly on primary articles published within the last few years. Primary articles are those which make the initial report of original research and have undergone a process of checking by other scientists that is called peer review. Primary reports contrast with secondary, or review reports, which make use of primary sources to discuss and summarize the current state of information in an area. They bring together many primary articles to present an overall, up-to-date picture of a topic. Secondary articles are often helpful in locating primary articles or defining a topic. Because it will be based upon primary sources, your term paper will ultimately resemble one of these review articles. You should consciously model your term paper on a review article such as those which appear in the “Annual Review” series, available electronically on the IUP campus.

Tertiary sources, such as textbooks, will be of little help in your work, since they are usually outdated by years at the time of publication

You will probably find that internet searches of the literature will make your job much easier. You will also probably find that there are many pages and sites that deal with different aspects of virology. You may use these sites for background information but the main thrust of your paper should deal with reports in the primary literature. Unlike web sites, these reports have been reviewed by peers and may be assumed to be of greater value and accuracy.

Web sites such as Wikipedia are not acceptable sources. Your citations must come from peer-reviewed materials.

You will be required to keep the instructor up-to-date on the progress of your literature review and you will be graded on how well you do so. See the Term Paper Schedule for details.

 

ALL STUDENTS WILL NEED TO DISCUSS THEIR REFERENCES WITH ME AND SHOW ME THE REPRINTS!!!!!

 

C: Organization of paper

The final paper must organized into the four main sections listed below. To organize your paper further, use subheadings wherever possible within each section.

(1) Introduction

(2) Body

(3) Conclusion

(4) Bibliography

 

(1) In the Introduction, briefly review and describe the viruses you will discuss as well as the biological property being reviewed (replication or transmission for example). The general state of knowledge in the area and the pertinence of the topic should also be described. Include in the Introduction a list of the abbreviations you will use in the term paper.

 

(2) In the Body of your paper you summarize the knowledge in the field, discussing the pertinent contributions of each important primary report, one at a time. In essence, you use the Body to present the results of your literature review. In this part of the paper you are getting your message across, explaining the material to your readers. It is your job as writer to organize, reorganize, synthesize, paraphrase, summarize----whatever it takes to package the information in a form which is easy for the reader to understand.

 

(3) The Conclusion does two things. First, it summarizes the results of your literature review. Second, it pulls together the information you have collected to produce a "take-home message". In other words it synthesizes the important information.

 

(4) Bibliography lists the primary, secondary and tertiary literature you have consulted. For literature citations, use the same format as that used by articles in the Journal of Virology. I expect all students, both graduate and undergraduate, to do a professional job of searching the literature and I expect their bibliographies to show evidence of this. Microscopic bibliographies containing many tertiary sources or only a half-dozen or so references will count against your grade. I fully realize the difficulties of attempting to do library work at IUP and I also realize that the only way to surmount them is to start soon and to keep with it. Waiting until the last minute guarantees extra problems and lower grades.

Papers not conforming to format guidelines will be returned ungraded.

You will be expected to keep the instructor posted as you progress through the stages of writing your paper. See the Term Paper Schedule for details.

 

D. Style

The paper is to be written in the general style of a regular scientific review article (see the library's electronic collection of "Annual Review" serials for examples). Write the paper in standard scientific English, using jargon where appropriate and with definitions. Use complete sentences with correct punctuation and minimal spelling errors. Assume that your audience consists of a group of upper-division, advanced undergraduate students of at least your own level of education. If there are questions of style, usage, spelling, abbreviations, format, etc. refer to the Journal of Virology for guidance. Papers must be typewritten or neatly handwritten. Papers not conforming to stylistic guidelines will be returned ungraded.

 

E. Schedule

The deadlines for submitting various stages of the term paper are given below. These deadlines are in place to ensure that students make regular and steady progress toward completion of the paper and to discourage procrastination. Except in cases of dire emergency (as judged by the instructor on a case-by-case basis), failure to meet a deadline will result in a grade of zero for the appropriate segment of the term paper.

March 1 (Monday) at 4:30 PM This is the deadline for turning in a preliminary term paper topic in hard copy format and discussing it with me (aka First Progress Report). These may be subsequently revised, if necessary. You have to have a topic and a few references at this point. We will also discuss your literature search.

April 5 (Monday) at 4:30 PM At this time a brief outline of the paper is due (aka Second Progress Report) in hard copy format. The major subheadings of Introduction, Body, and Conclusion must be in place. The important points in each subheading should be listed. The bibliography should be well advanced, with all of the key references and most of the others listed. Subsequent revisions are permitted. We will have a more in-depth discussion of your literature search at this meeting.

May 3 (Monday) Finished papers due at 4:30 PM.

If you have any questions about the format or other requirements for brief outline, preliminary bibliography, and so on, feel free to contact me for clarification.

 

F. TERM PAPER GRADING

Your paper will be graded on a 100-pt. scale with points awarded according to the following schedule.

Source Points Due Date

Brief Outline/

First Progress Report 5 Monday March 1 at 4:30 PM

Final Outline/

Second Progress Report 5 Monday April 5 at 4:30 PM

Finished Paper 90 Monday May 4 at 4:30 PM

You will receive full credit for the term paper topic, the brief outline and the final outline as long as you turn them in ON TIME and written according to the guidelines established above.

Finished papers will be assigned first a letter grade and then a number grade from the range corresponding to the letter. Comments will be written on each individual paper Letter grades and their corresponding number range are given below.

 

Letter

%

Range

Comment

A

>92-100

Excellent; you conducted a thorough survey of the literature, concisely synthesized the data, communicated the results effectively, summarized and produced a take-home-message, used correct scientific terminology and followed the format

B

>82-92

Above Average; you did many but not all of the above; see paper for more comments

C

>72-82

Average; you did some but not all of the above; see paper for more comments

D

>62-72

Below Average; you did not do most of the above; see paper for more comments

F

<62

Far Below Average; you did few or none of the above, did not turn in a paper, or did not meet a deadline

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UK POLICY FOR MANAGEMENT OF POTENTIAL ORGAN/TISSUE DONORS WITH CONFIRMED POSITIVE VIROLOGY RESULTS (UPDATE)

1. INTRODUCTION

1.1 This policy provides guidance on the management of potential organ/tissue donors with positive virology results. It will give healthcare professionals guidance on how to proceed when donation cannot take place as a consequence in light of these results, the subsequent care of the potential donor, and their next of kin/significant other.

1.2 Transplantation is now well established, as the treatment of choice for the majority of patients with end stage organ failure. However, the transmission of infection is one of the associated risks. All potential organ/tissue donors have blood taken for a number of virology tests. Please refer to the Microbiological Safety of Blood and Tissues for Transplantation (MSBT) guidelines.1 In addition, the donor/tissue transplant co-ordinator is required to take a detailed medical and social history.

1.3 When organ donation is discussed with the family of the potential donor, and consent for donation is agreed, this process also informs the donor’s family that tests for a the consent will include testing for a number of possible viral infections. will be carried out on the blood of the potential donor. Moreover, the consent form also says that in the event of a confirmed positive result becoming available that may have clinical relevance to any family member, then the result and its implications will be discussed with them.

1.4 Confidentiality is one of the most significant concepts in healthcare and is an important factor in client/practitioner relationships as stated in the codes of conduct (General Medical Council (GMC) 19932 /Nursing Midwifery Council 2002).3

1.5 Under The NHS (Venereal Diseases) Regulation 19744 and the NHS Trusts (Venereal Diseases) Directions 19915 it:

“prevents the disclosure of any identifying information about a patient examined or treated for a sexually transmitted disease (including HIV & AIDS) other than to a medical practitioner (or to a person employed under the direction of a medical practitioner) in connection with and for the purpose of whether the treatment of the patient and/or the prevention of the spread of the disease.”

This regulation demonstrates that there are statutory duties to ensure all steps are taken to prevent any information, that is capable of identifying a patient with a sexually transmitted disease being disclosed. However the disclosure of this information could arguably be possible for the purposes of treatment and prevention of the spread of the disease to partners and others who may be affected as a result of their relationship to the potential organ donor.

1.6 This Policy has been devised to provide guidelines, information and support for healthcare professionals where positive virology results have been confirmed on potential organ/tissue donors and where these results have implications for others. It is important that this is dealt with in an appropriate manner pertinent to the merit of each individual case.

 

2. REVIEW

2.1 This document will be reviewed every two years or as required.

 

3. AIM

3.1 To identify all potential donors with positive virology

3.2 To determine the necessity for disclosure of this information to the next of kin/significant other.

3.3 To obtain expert advice.

3.4 To repeat and confirm virology results.

3.5 To determine who discloses this information.

3.6 To disclose the findings with the appropriate healthcare specialists.

3.7 To ensure that any discussions with the next of kin/significant other are appropriately documented in the potential organ/tissue donor’s medical notes.

3.8 To ensure documentation of reasons for disclosure are clear and concise including contact/advise given from appropriate individual healthcare professionals.

3.9 Disclosure to take place within an appropriate time scale.

3.10 To advise the next of kin/significant other of support networks available.

 

4. RATIONALE

4.1 It is acknowledged that in the UK there is no current legislation regarding the breach of confidentiality when disclosing positive virology results of potential cadaver donors (especially when a third party health is at risk). In contrast there are many states in America where there is a legal obligation to disclose confidential information if there is a third party who may be affected (Cochran 1999).6

4.2 The British Medical Association (BMA) Confidentiality and Disclosure of Health Information document (1999)7 states:

‘post mortem test of a cadaver may reveal the presence of previously undiagnosed infectious conditions which people close to the deceased person may need to be aware of in order to protect their own health or that of others’

4.3 The ethos of the Department of Health (DOH), BMA and GMC is that the decision to disclose information regarding positive virology results should be made by balancing the benefits with the harm of the public interest. This includes the legitimate concern for the interests of others who may be affected by the person with the virus. The GMC (1993) has concluded:

‘there are grounds for such disclosure only where there is a serious and identifiable risk to a specific individual who, if not so informed, would be exposed to the infection………………………but where such consent is withheld the doctor may consider it a duty to seek to ensure that any sexual partner is informed, in order to safeguard such persons from a possible fatal infection’ (GMC cited in Kennedy, Chapter 9, pg 6648).

4.4 The DOH issued guidance on partner notification for HIV in December 19929 and stated that the benefits of notification are the following:

q Identified contacts are given the opportunity to consider whether they wish to be tested

q Those who have unknowingly been infected may wish to take steps to prevent transmission to others

q Access to treatment and support programmes so that they may benefit from long term monitoring of their clinical condition and from appropriate therapies.

4.5 Although the above guidance has only stipulated the disclosure for HIV infection, Hepatitis B and C are important viruses and the implications of such infections can be equally as life threatening as HIV. These guidelines therefore may be used to address all positive virology results where there are implications for a third party.

4.6 Informing potentially infected individuals may have various implications. However, advances in medical treatments, therapies and support networks may balance the benefit and outweigh the harm for most individuals.

4.7 A nationwide policy will give guidance to the healthcare professionals in dealing with this ethically and morally difficult issue.

 

5. HIV

5.1 When a potential organ/tissue donor has been identified as HIV positive this information will be relayed to the duty donor/tissue transplant co-ordinator.

5.2 The donor/tissue transplant co-ordinator will stop the donation process, informing all the relevant parties but maintaining the need for confidentiality.

5.3 The result will be reconfirmed by the laboratory medical staff prior to disclosure.

 

6. HEPATITIS B/C

6.1 When a potential organ/tissue donor has been identified as hepatitis positive this information will be relayed to the donor/tissue transplant co-ordinator.

6.2 The donor/tissue transplant co-ordinator will inform UKT and the relevant retrieval teams of the result.

6.3 If the retrieval teams decline the offer the donor transplant co-ordinator will inform UKT. The organs will continue to be offered in accordance with national guidance.

6.4 This result will be reconfirmed by the laboratory medical staff prior to disclosure

 

7. SYPHILIS

7.1 On obtaining a positive syphilis result the donor/tissue transplant co-ordinator will inform the transplant team and UKT, as appropriate.

7.2 This result will be reconfirmed by the laboratory medical staff prior to disclosure.

 

8. NOTIFICATION OF NEXT OF KIN/SIGNIFICANT OTHERS

8.1 The donor/tissue transplant co-ordinator will discuss with the clinician in charge of the potential donor an action plan on what information is to be disclosed to the next of kin/significant other at this early stage and how this information will be disclosed and by whom.

8.2 An appointment will be arranged to discuss results with the next of kin/significant other, however, this should take place after the confirmatory results have been received and expert advice has been obtained.

8.3 The clinician in charge of the care of the potential donor should be involved in this meeting and it may be appropriate to include an expert practitioner within this field. This meeting should include no less than two appropriate healthcare professionals. Advice may be obtained from the hospital legal department and the medical director of UKT before any meeting takes place if necessary.

8.4 The information to be discussed at this meeting should include:

  • A detailed discussion of the positive result and the implications to the next of kin/significant other
  • An offer to screen all those who may be at risk
  • The need for precautions to be taken until their results are confirmed. If they decline to be tested then advice should be given on prevention of transmission.
  • Approval should be sought and encouraged for permission to divulge these findings to their General Practitioner.
  • Contact numbers for local clinics and support groups/counsellors should be given to the next of kin/significant other.

 

8.5 Detailed records of all conversations should be entered into the notes of the potential donor/ donor medical records.

8.6 Letter should be sent to the General Practitioner of the potential donor/donor.

 

REFERENCES

1 Department of Health, Advisory Committee on the Microbiological Safety of Blood and Tissues for Transplantation.Guidance on the Microbiological Safety of Human Organs, Tissues and Cells used in Transplantation (August 2000).

2 General Medical Council (1993) Confidentiality: Protecting and Providing Information

3 Nursing and Midwifery Council (2002) Code of Professional Conduct

4 The National Health Service (Venereal Diseases) Regulation 1974, No 29

5 The National Health Service Trusts (Venereal Diseases) Directions 1991

6 Cochran M (1999) The real Meaning of Patient-Nurse Confidentiality, Critical Care Nurse Quarterly 22 (1) pp 42-51

7 British Medical Association (1999) Confidentiality and Disclosure of Health Information

8 Kennedy I, Grubb A (1994) Medical Law, Chapter 9, pg 664, 2nd Edition, Butterworth & Co Ltd, London

9 Department of Health (1992) PLICO (92) 5 Guidance on Partner Notification for HIV Infection

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