Differentiation of junin virus and antigenic variants isolated in vivo by kinetic neutralization assays

The major natural reservoir of Junin virus, the aetiological agent of Argentine haemorrhagic fever, is the cricetid Calomys musculinus. Neonatal animals experimentally infected with Junin virus (XJCl3 strain) developed typical disease and approximately 80% of them died. Most survivors become persistently infected. Antigenically variant viruses were isolated from the blood and brain of infected cricetids during the acute and chronic stages of the disease. These variants could be distinguished from the parental strain by kinetic neutralization assays using polyclonal antibodies. Some biological properties were shared with the parental virus strain including its virulence for newborn C. musculinus. These variant viruses may play a major role in chronic disease since we have shown that a viral isolate from an infected brain was poorly neutralized by serum obtained from the same animal.Fil:Alche, L.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Coto, C.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Characterization of the Influences of Human Cytomegalovirus Glycoprotein O (gO) Expression on gH/gL Complexes Assembly and Its Polymorphisms on Cell-free and Cell-to-cell Spread, and Antibody Neutralization.

Human cytomegalovirus (HCMV) is widely spread throughout the world and immunocompromised individuals can suffer severe diseases from HCMV infection. Once the infection is established, HCMV can spread through the body and infect many major somatic cell types. The glycoproteins H and L (gH/gL) on HCMV envelope can be bound by either gO or the UL128-131 proteins to form complexes gH/gL/gO and gH/gL/UL128-131 that are critical for viral entry and spread, and these two complexes are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131. gO is one of the most diverse loci among strains with 10-30% of amino acid sequence differences. In this thesis I explored the mechanisms behind the complex assembly differences between strains and the impacts of interstrain gO diversity on the biology of HCMV. My results uncovered that the strain variations in the assembly of gH/gL complexes is due to the differences in the expression level of gO and UL128-131, while gO amino acid sequence differences have no influence on the complexes assembly. Interestingly, the diversity of gO has dramatic impacts on HCMV cell-free and cell-to-cell spread as well as on antibody neutralization and these effects of gO polymorphisms are epistatically dependent on other variable loci in the virus genome. My study could help to understand the complexity of genotypes observed in clinical samples and decode the challenge for intervention approaches against HCMV

Die Rolle des Polymorphismus des Glykoproteins N des humanen Cytomegalovirus bei der humoralen Immunantwort und der Virusreplikation

Human cytomegalovirus is a ubiquitous human herpes virus which, after a primary infection, persists lifelong in its host. CMV infection causes no or only mild symptoms in immunocompe-tent persons, but can lead to life-threatening disease in immunosuppressed persons, such as graft recipients, neonates or AIDS patients. Reinfections with other CMV strains can occur despite existing immunity. To design an effective viral vaccine, the interactions between virus and host need to be understood in detail, in particular the mechanisms of development of a humoral im-mune response and determination of cell tropism of the virus. The envelope of HCMV comprises several proteins that differ drastically in primary amino acid sequence among viral strains. The glycoprotein (g) N exhibits a variability of up to 50 % and is the most polymorphic protein of the virus envelope. gN proteins can be divided in four groups (gN1, gN2, gN3, gN4) with further subdivision (gN3a, gN3b, gN4a, gN4b, gN4c). In this work the influence of the polymorphism of gN on the humoral immune response and virus replication in different cell types was investi-gated. It was demonstrated that the gN subtypes elicit the production of subtyp-specific antibodies du-ring natural infection. For this purpose four recombinant viruses with different gN subtypes were constructed in the genetic background of the HCMV strain AD169. The recombinant viruses were tested for neutralisation by sera of HCMV-seropositive persons in an in vitro neutralisation assay. Among the 20 serum samples tested two types of sera were identified: strain-common sera (14) that neutralised all four viruses equally well and strain-specific sera (6) that exhibited diffe-rences in the neutralisation titers against the four recombinant gN-viruses. The differences in neutralisation titers of strain-specific sera reached up to sevenfold and were detectable despite the activity of other, so far considered dominant, neutralising antibodies, e.g. against gB or gH. The gN4-bearing virus was significantly better neutralised than the other gN-viruses. The sera were tested in an ELISA for antibodies against transiently expressed gN. Five of the strain-specific sera displayed considerably low titers against gN2 than against the other gN subtypes. Three of the strain-common sera also showed notably low gN2-titers, one serum exhibited no gN4-titer, but high titers against all other gN subtypes. All other strain-common sera displayed comparable titers against the gN subtypes. With respect to gN neutralisation titers and ELISA titers did not correlate. To identify binding sites of gN subtyp-specific antibodies, recombinant viruses with truncated gN proteins were generated. However, in spite of the absence of an immunogenic part of gN the recombinant viruses were equally well or better neutralised in in vitro neutralisation assays than the respective virus containing the entire gN. Thus, an identification of antibody-binding epi-topes on gN was not feasible with this approach. As a viral envelope protein gN subtypes can theoretically play a role in virus replication in dif-ferent cell types, for example by influencing virus attachment and/or penetration. This assump-tion was tested in a replication assay in fibroblasts and endothelial cells under condition of com-petition between the recombinant gN-viruses. For infection of endothelial cells four recombinant viruses with different gN subtypes were constructed in the genetic background of the endothelio-tropic HCMV-isolate TR. Cell cultures of fibroblasts and endothelial cells were infected with the corresponding four recombinant viruses. One day after infection and again after 10 passages of the infection the ratio of the four viral genomes was determined. After 10 passages of the infec-tion the genome of the gN2-virus was 20-fold less present than the genomes of the other gN-viruses. After 10 passages of the infection in endothelial cells the genome of the gN4-virus was no longer detectable, whereas the other genomes were present in equal ratios. The results of this work demonstrate that the polymorphism of gN induces the formation of a strain-specific immune response and thus could contribute to the well known phenomenon of reinfection of immune hosts. The protein could also influence the viral replication in different cell types and thus determine viral spread in vivo or even pathogenesis.Das humane Cytomegalovirus ist ein ubiquitär vorkommendes -Herpesvirus, welches nach In-fektion lebenslang im Wirt persistiert. In immunkompetenten Personen verläuft eine Primärin-fektion meist asymptomatisch, in immunsupprimierten Personen, wie z.B. Transplantationspa-tienten, Neugeborenen oder AIDS-Patienten, können jedoch schwerwiegende Komplikationen auftreten. Reinfektionen mit anderen HCMV-Stämmen können trotz vorhandener Immunität auf-treten und bei immunsupprimierten Personen zu symptomatischen Krankheitsverläufen führen. Für die Entwicklung einer effektiven Prophylaxe ist es notwendig, die Interaktionen zwischen Virus und Wirt detailliert zu verstehen. Von elementarem Interesse sind dabei die Mechanismen der Entwicklung einer humoralen Immunantwort gegen das infizierende Virus und die Determi-nation des Zelltropismus des Virusstammes. HCMV besitzt in seiner Hülle mehrere Hüllprotei-ne, die unter verschiedenen Virusstämmen in ihrer Struktur variieren. Das Glykoprotein (g) N ist mit ca. 50 % Variabilität das polymorphste Protein der Virushülle. gN-Proteine werden mit zu-sätzlichen Untergliederungen in vier Subtypklassen (gN1, gN2, gN3a, gN3b, gN4a, gN4b, gN4c) eingeteilt. In der vorliegenden Arbeit wurde die Rolle des Polymorphismus von gN bei der hu-moralen Immunantwort und bei der Virusreplikation in verschiedenen Zelltypen untersucht. Es konnte nachgewiesen werden, daß die gN-Subtypen während einer natürlichen Infektion die Bildung von gN-subtypspezifischen Antikörpern hervorrufen. Dazu wurden vier rekombinante Viren im genetischen Hintergrund des HCMV-Stammes AD169 mit unterschiedlichen gN-Subtypen generiert, die den Subtypklassen gN1, gN2, gN3 bzw. gN4 zugeordnet werden. Die rekombinanten gN-Subtyp-Viren wurden in einem in vitro Neutralisationstest auf Neutralisation durch Seren HCMV-seropositiver Personen getestet. Von 20 untersuchten Seren wiesen 14 Seren gleiche Neutralisationstiter gegen die vier gN-Subtyp-Viren auf und wurden stammübergreifend genannt. Sechs Seren neutralisierten die gN-Subtyp-Viren mit unterschiedlichen Titern und wur-den stammspezifisch genannt. Die Unterschiede in den 50 %-Neutralisationstitern der stamm-spezifischen Seren betrugen bis zu siebenfach und waren trotz Aktivität anderer neutralisierender Antikörper, z.B. gegen gB und gH, detektierbar. Innerhalb der Stichprobe von 20 Seren wurde das gN4-tragende Virus signifikant besser neutralisiert als Viren, die andere gN-Subtypen in ih-rer Hülle trugen. Die Seren wurden im ELISA auf Antikörper gegen transient exprimiertes gN getestet. Fünf der stammspezifischen Seren wiesen beträchtlich niedrigere Titer gegen gN2 als gegen die restlichen gN-Subtypen auf. Drei der stammübergreifenden Seren zeigten ebenfalls beträchtlich niedrigere Titer gegen gN2 als gegen die anderen gN-Subtypen, ein Serum wies kei-nen gN4-Titer, aber hohe Titer gegen die restlichen gN-Subtypen auf. Alle anderen stammüber-greifenden Seren zeigten vergleichbar hohe Titer gegen die vier gN-Subtypen. Zwischen den Neutralisations- und den ELISA-Titern konnte keine Korrelation festgestellt werden. Zur näheren Bestimmung der Bindestellen gN-subtypspezifischer Antikörper wurden rekombi-nante Viren mit verkürzten gN-Proteinen generiert. In in vitro Neutralisationstests wurden diese Viren trotz Fehlen eines immunogenen Teils von gN gleich gut oder besser neutralisiert als das entsprechende Virus mit vollständigem gN. Eine Identifizierung von neutralisationsrelevanten Epitopen war somit nicht möglich. Als virale Hüllproteine können gN-Subtypen theoretisch auch bei der Replikation in verschiede-nen Zelltypen eine Rolle spielen, z.B. durch Beeinflussung des Andockens des Virus an die Zelle oder der Penetration in die Zelle. Dies wurde in einem Versuch zur Virusreplikation unter Kon-kurrenzbedingungen in Fibroblasten und Endothelzellen untersucht. Für die Infektion von Endo-thelzellen wurden vorher analog zu den RVAD169-Viren vier rekombinante Viren mit geneti-schem Hintergrund des endotheliotropen HCMV-Isolates TR generiert, die jeweils einen der vier gN-Subtypen in ihrer Hülle trugen. Fibroblasten- bzw. Endothelzellkulturen wurden mit allen vier RVAD169- bzw. RVTR-Viren gleichzeitig infiziert. Aus den infizierten Zellkulturen wur-den einen Tag nach Infektion und nach 10-maliger Passagierung des Infektes die Verhältnisse der vier Virusgenome bestimmt. In der Fibroblastenkultur lagen nach 10-maliger Passagierung die Genome des gN2-Virus bis zu 20 mal weniger vor, als die der anderen gN-Subtyp-Viren. In der Endothelzellkultur war nach 10-maliger Passagierung das Genom des gN4-Virus nicht mehr nachweisbar. Alle anderen Virusgenome lagen in vergleichbaren Verhältnissen vor. Die Ergebnisse dieser Arbeit zeigen, daß der Polymorphismus von gN die Induktion einer Virus-stamm-spezifischen humoralen Immunantwort auslöst und damit zum bekannten Phänomen der Reinfektion beiträgt. Der Polymorphismus von gN beeinflußt auch die Virusreplikation in unter-schiedlichen Zelltypen und könnte sich dadurch auf die Ausbreitung des Virus in vivo oder sogar auf die Pathogenese von HCMV auswirken

Viral and host gene expression during human cytomegalovirus infection.

HCMV infection is usually asymptomatic in immunocompetent hosts, but serious disease can occur in immunocompromised individuals and in congenitally infected newborns. The importance of virus fitness determinations became evident in 1976, when it was proposed that the infecting strain of HCMV is important for clinical outcome, along with the intensity and duration of viral replication. HCMV strains exhibit different levels of virulence in vivo, depending on their passage history in cell culture. High and low passage HCMV strains exhibit tropism differences in vitro, suggesting that different tissue tropism may occur in vivo. In addition, approximately 13kb of novel DNA sequences located near the right edge of the unique long component of the genome has been identified in Toledo and clinical strains. This region (UL/b') encodes several open reading frames, which are missing from the high passage laboratory-adapted variants of Towne and AD 169 and are thought to play important roles in pathogenesis. One of the aims of this thesis was to determine the replication dynamics of different HCMV strains in vitro as well as compare their ability to bind to cells and mediate cell-to-cell spread of infection using pair-wise competition experiments in cell culture. AD 169 was shown to replicate better than Toledo in fibroblasts. Furthermore, assessment of the replication of Toledo in a different cell type, HUVEC, indicated that the virus replicated to higher levels in fibroblasts. Towne was found to bind to HEL cells with higher affinity compared to AD 169. The results showed phenotypic differences between high and low passaged HCMV variants and also illustrated that fitness differences between them are variable and highly dependent upon the status of the virus inoculum. To begin to understand the complex relationship between tissue tropism, virulence and HCMV genome composition, a DNA microarray approach was developed to examine host and HCMV gene expression during the productive infection of two distinct cell lines, fibroblasts and endothelial cells. The results showed that genes wifrojn tye fPSIPp were expressed in a cell type-specific fashion. Ip the context of host cell gene expression, cell type-specific host gene transcriptional changes were observed, reflecting different viral modulation of distinct cell type environments. The results provided potential insight into the function of genes encoded in the UL/b' region. Of particular note was that transcriptional changes frequently occurred in genes associated with pathways involved in the pathology of HCMV in the human host