Catherine A. Laughlin

Cross-Species Virus Transmission and the Emergence of New Epidemic Diseases

SUMMARY Host range is a viral property reflecting natural hosts that are infected either as part of a principal transmission cycle or, less commonly, as “spillover” infections into alternative hosts. Rarely, viruses gain the ability to spread efficiently within a new host that was not previously exposed or susceptible. These transfers involve either increased exposure or the acquisition of variations that allow them to overcome barriers to infection of the new hosts. In these cases, devastating outbreaks can result. Steps involved in transfers of viruses to new hosts include contact between the virus and the host, infection of an initial individual leading to amplification and an outbreak, and the generation within the original or new host of viral variants that have the ability to spread efficiently between individuals in populations of the new host. Here we review what is known about host switching leading to viral emergence from known examples, considering the evolutionary mechanisms, virus-host interactions, host range barriers to infection, and processes that allow efficient host-to-host transmission in the new host population.

Cloning of infectious adeno-associated virus genomes in bacterial plasmids

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

Adeno-associated virus autointerference

We have analyzed an autointerference phenomenon exhibited by adeno-associated virus type 2 (AAV) when grown in KB cells coinfected with adenovirus type 2 as the helper. Infectious AAV particles that banded at 1.41 g/cm3 in CsCl were purified by three cycles of centrifuging in CsCl equilibrium gradients. When cells were infected with an increasing multiplicity of these AAV particles there was a corresponding decrease in production of infectious progeny AAV. There was also an AAV multiplicity-dependent inhibition of production of infectious adenovirus and inhibition of Ad DNA replication. The viral DNA in the Hirt supernatant fraction extracted from cells infected with different multiplicities of AAV was analyzed in neutral sucrose gradients. At low multiplicities of infection with AAV, the main AAV DNA species synthesized was the mature 14.5 S (standard) viral genome. In higher multiplicity infections with AAV increasing amounts of aberrant 10 S AAV DNA molecules accumulated and the proportion of 14.5 S AAV DNA decreased. Restriction endonuclease cleavage showed that the 10 S DNA was enriched for the left- or right-hand terminal regions of the AAV genome. These molecules may be analogous to the previously characterized aberrant DNA molecules found in light-density AAV particles. Thus, the AAV autointerference is correlated with production of the aberrant deleted AAV genomes.

Priorities for CMV vaccine development.

A multidisciplinary meeting addressed priorities related to development of vaccines against cytomegalovirus (CMV), the cause of congenital CMV (cCMV) disease and of serious disease in the immunocompromised. Participants discussed optimal uses of a CMV vaccine, aspects of clinical study design, and the value of additional research. A universal childhood CMV vaccine could potentially rapidly reduce cCMV disease, as infected children are sources of viral transmission to seronegative and seropositive mothers. A vaccine administered to adolescents or adult women could also reduce cCMV disease by making them immune prior to pregnancy. Clinical trials of CMV vaccines in women should evaluate protection against cCMV infection, an essential precursor of cCMV disease, which is a more practical and acceptable endpoint for assessing vaccine effects on maternal-fetal transmission. Clinical trials of vaccines to evaluate prevention of CMV disease in stem cell transplant recipients could use CMV viremia at a level triggering pre-emptive antiviral therapy as an endpoint, because widespread use of pre-emptive and prophylactic antivirals has rendered CMV-induced disease too rare to be a practical endpoint for clinical trials. In solid organ transplant patients, CMV-associated disease is sufficiently common for use as a primary endpoint. Additional research to advance CMV vaccine development should include identifying factors that predict fetal loss due to CMV, determining age-specific incidence and transmission rates, defining the mechanism and relative contributions of maternal reactivation and re-infection to cCMV disease, developing assays that can distinguish between reactivation and re-infection in seropositive vaccinees, further defining predictors of sequelae from cCMV infection, and identifying clinically relevant immune response parameters to CMV (including developing validated assays that could assess CMV antibody avidity) that could lead to the establishment of immune correlates of protection.

Report of an NIAID workshop on dengue animal models.

Dengue is a mosquito-borne viral disease of humans that has re-emerged in many parts of the world and has become an important international public health threat. Dengue incidence and geographical spread has dramatically increased in the last few decades and is now affecting most tropical and sub-tropical regions of the world. Despite extensive research efforts for several decades, no vaccines or therapeutics are currently available to prevent and treat dengue infections. One of the main obstacles to the development of countermeasures has been the lack of good animal models that recapitulate dengue pathogenesis in humans and reliably predict the safety and efficacy of countermeasures against dengue. In September 2008, the National Institute of Allergy and Infectious Diseases (NIAID) held a workshop to consider the current state-of-the-art developments in animal models for dengue and discuss strategies to accelerate progress in this field. This report summarizes the main discussions and recommendations that resulted from the meeting.

Dengue Research Opportunities in the Americas

Dengue is a systemic arthropod-borne viral disease of major global public health importance. At least 2.5 billion people who live in areas of the world where dengue occurs are at risk of developing dengue fever (DF) and its severe complications, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Repeated reemergences of dengue in sudden explosive epidemics often cause public alarm and seriously stress healthcare systems. The control of dengue is further challenged by the lack of effective therapies, vaccines, and point-of-care diagnostics. Despite years of study, even its pathogenic mechanisms are poorly understood. This article discusses recent advances in dengue research and identifies challenging gaps in research on dengue clinical evaluation, diagnostics, epidemiology, immunology, therapeutics, vaccinology/clinical trials research, vector biology, and vector ecology. Although dengue is a major global tropical pathogen, epidemiologic and disease control considerations in this article emphasize dengue in the Americas.

Defective-interfering particles of the human parvovirus adeno-associated virus

Abstract We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.450, AAV(1.41), AAV(1.35), and AAV(1.32), respectively, where the density of the particles was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV particles. These infectious AAV particles exhibited autointerference. The light-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious but interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5 S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.

Properties of an adenovirus type 2 mutant, Ad2d/807, having a deletion near the right-hand genome terminus: Failure to help AAV replication

We have analyzed the ability of an adenovirus type 2 mutant, Ad2dl807, to support replication of adeno-associated virus (AAV). This mutant has a deletion extending from early region 3 through the late fiber gene and into early region 4. Since AAV growth does not require Ad early region 3 or the fiber gene, the Ad2dl807 mutant allows analysis of the function of Ad early region 4 in AAV growth. As determined by assay of AAV DNA, RNA, and protein synthesis as well as the number of cells producing AAV and the yield of infectious AAV particles, growth of AAV with the dl807 mutant is decreased 10- to 20-fold. The residual AAV growth is probably due to the presence of a low level of contaminating viable Ad virions in the dl807 preparations. These observations indicate that an Ad early region 4 function is required at a very early stage of AAV DNA replication to allow amplification of duplex replicating (RF) DNA. Thus growth of AAV offers an independent probe for adenovirus early region 4 functions.

NIAID resources for developing new therapies for severe viral infections

Abstract Severe viral infections, including hemorrhagic fever and encephalitis, occur throughout the world, but are most prevalent in developing areas that are most vulnerable to infectious diseases. Some of these can also infect related species as illustrated by the threatened extinction of gorillas by Ebola infection in west and central Africa. There are no safe and effective treatments available for these serious infections. In addition to the logistical difficulties inherent in developing a drug for infections that are sporadic and occur mainly in the third world, there is the overwhelming barrier of no hope for return on investment to encourage the pharmaceutical industry to address these unmet medical needs. Therefore, the National Institute of Allergy and infectious Disease (NIAID) has developed and supported a variety of programs and resources to provide assistance and lower the barrier for those who undertake these difficult challenges. The primary programs relevant to the development of therapies for severe viral infections are described and three case studies illustrate how they have been used. In addition, contact information for accessing these resources is supplied.

Adeno-Associated Virus Defectiveness and the Nature of the Adenovirus Helper Function

Adeno-associated virus (AAV) was first observed as a contaminant in preparations of adenovirus (Brandon and Maclean, 1962; Archetti and Bocciarelli, 1963, 1964, 1965; Archetti et al., 1965; Hoggan, 1965; Hull et al., 1965; Melnick et al., 1965) and subsequently recognized as a defective virus which was unconditionally dependent for its replication upon coinfection of the host cell with a helper adenovirus (Atchison et al., 1965; Hoggan et al.,1966; Parks et al., 1966). It was also realized that one other group of viruses, namely, herpes viruses could provide helper functions for the production of AAV DNA, RNA, and proteins (Atchison, 1970; Blacklow et al., 1970; 1971a; Boucher et al., 1971; Carter and Rose, 1972; Carter et al., 1972; Dolin and Rabson, 1973; Rose and Koczot, 1972). For some time, it was generally believed that the herpes virus helper function for AAV was incomplete and that either formation of AAV capsids or packaging of AAV DNA into capsids failed to occur (Atchison, 1970; Henry et al.,1972). However, more recent work has demonstrated that herpes viruses can provide a complete AAV helper function and allow production of fully infectious AAV particles (Buller et al., 1981).