Mathias Ackermann

Pathogenesis of ruminant herpesvirus infections

Ruminants are hosts for members of both Alpha- and Gamma-herpesvirinae. A wide range of disease syndromes is associated with infections by these agents. The associated diseases reflect the biological nature of the causative viruses. Clinically, the symptoms may be mild and localized or include severe generalized disease, leading eventually to death. Much knowledge has been gained concerning the pathogenesis of some alpha-herpesviruses. Initially, these viruses replicate in epithelial cells at the portal of entry. The symptoms of the acute diseases are often associated with the destruction of those epithelial cells. However, as in the case of bovine herpesvirus 1 (BHV-1), the virus may spread in the infected host by viremia, gaining access to a broader range of tissues and organs, and causing a broader variety of diseases. Furthermore, many herpesviruses are capable of entering neuronal cells. There, they may replicate, which may lead to neuronal diseases, for example, encephalitis. In addition, the herpesviruses may establish latency in neuronal or lymphoid cells. During latency, apparently no viral antigens are synthesized but the genomes of the latent viruses are present in the nuclei of long living cells, such as, e.g., neurones of the ganglia corresponding to the sites of peripheral replication. Upon reactivation, the viruses re-establish the lytic cycle of replication. Shielded from the effectors of the immune system, they migrate back to the peripheral tissues where they are excreted and may be transmitted. Although a strong immune response is provoked during primary viral replication, these mechanisms help the herpesviruses to escape from immune surveillance during latency and to a lesser degree during reactivation. It has been observed that certain herpesviruses may behave differently upon infection of different hosts. Relatively little progress has been made concerning the understanding of the pathogenesis of ruminant herpesviruses but much has been learned about viral molecular biology. Many viral proteins have been identified and characterized and the technology to create recombinant viruses has been established. With these tools in our hands, it is now possible to address the really interesting questions concerning pathogenesis. We postulate that herpesviruses contain at least two sets of genes, a first set involved in gene expression and viral replication, and a second set responsible for functions, which may affect pathogenesis, latency, and virus/host interactions. Using recombinant virus technology, it will be possible in the future to design targeted deletions and gene transfers in ruminant herpesviruses in order to study the viral and host factors involved in pathogenesis on the molecular level.

Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence.

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5′ end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.

Molecular virology of ruminant herpesviruses.

Molecular virology has served to establish bovine herpesvirus 1 (BHV-1) as the prototype member of ruminant herpesviruses. Based on the genomic sequence of the virus, we aim to identify and characterize virus-specified components, to explain their concerted action, and to predict how the chain of events during the lytic and latent phases of the viral life cycle may be interrupted. The nucleotide sequence of the BHV-1 genome (136 kb) has just been completed by international cooperation (July 1995; except for a small gap in UL36). It comprises 67 unique genes and 2 genes, both duplicated, in the inverted repeats. In general, these genes exhibit strong homology at the amino acid sequence level to those of other alphaherpesviruses (HSV-1, VZV, EHV-1) and are arranged in similar order. A few genes are peculiar to only one or two herpesviruses, e.g. in BHV-1 the circ, UL0.5, UL3.5 and US1.5 genes. Not long ago, the repertoire of BHV-1 proteins under study was restricted to the three major glycoproteins (gB, gC, and gD) and thymidine kinase. The repertoire is now growing rapidly and includes 7 additional glycoproteins (gE, gI, gH, gL, gG, gK and gM), a number of enzymes (e.g. ribonucleotide reductase, DNA Polymerase, dUTPase), and a group of regulatory proteins (BICPO, 4, 22, and 27, alpha TIF). Investigations into the functions of these proteins and comparison with their counterparts in other herpesviruses should reveal which are useful targets for diagnosis, prevention or antiviral treatment. Recombinant viruses containing deletions or replacements of individual genes are being created, aiming at vaccine development and insights into pathogenesis, notably latency, neurotropism, and interference with host functions. Molecular analysis of other ruminant herpesviruses is much less advanced. Over a dozen virus species have been described; most share basic properties with BHV-1 and may be classified as alphaherpesviruses. The gammaherpesviruses are represented by the proposed agent of malignant catarrhal fever, alcelaphine herpesvirus 1, and by bovine herpesvirus 4, whose partial sequences exhibit similarity to herpesvirus saimiri.

Herpes Simplex Virus 1 Envelopment Follows Two Diverse Pathways

ABSTRACT Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.

Quantitative Fluorogenic PCR Assay for Measuring Ovine Herpesvirus 2 Replication in Sheep

ABSTRACT A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.

Impairment of Nuclear Pores in Bovine Herpesvirus 1-Infected MDBK Cells

ABSTRACT Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.

Quantification of Feline Herpesvirus 1 DNA in Ocular Fluid Samples of Clinically Diseased Cats by Real-Time TaqMan PCR

ABSTRACT A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R2 = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

Interplay between Alpha/Beta and Gamma Interferons with B, T, and Natural Killer Cells in the Defense against Herpes Simplex Virus Type 1

ABSTRACT The essential components of the immune system that control primary and chronic infection with herpes simplex virus type 1 (HSV-1) in mice were investigated. Infection within the first few days can be controlled by alpha/beta interferon (IFN-α/β) alone without significant contribution of B, T, or NK cells. IFN-α/β and IFN-γ cooperate in the elimination of virus in the absence of these lymphocytes. In contrast, B, T, or NK cells appear to be required to control persistent infection with HSV-1. These results suggest that distinct and essential immune elements are recruited in a time-dependent fashion to control acute and persistent HSV-1 infection.

Flt3 Ligand–treated Neonatal Mice Have Increased Innate Immunity Against Intracellular Pathogens and Efficiently Control Virus Infections

Flt-3 ligand (FL), a hematopoetic growth factor, increases the number of dendritic cells (DCs), B cells, and natural killer cells in adult mice but the effect in neonates was unknown. We show that FL treatment of newborn mice induced a >100-fold increase in the innate resistance against infection with herpes simplex virus type 1 and Listeria monocytogenes. This resistance required interferon (IFN)-α/β for viral and interleukin (IL)-12 for bacterial infections. Long-term survival after viral but not bacterial infection was increased ∼100-fold by FL treatment. After treatment, CD11c+/major histocompatibility complex type II+ and CD11c+/B220+ DC lineage cells were the only cell populations increased in the spleen, liver, peritoneum, and skin. DC induction was independent of IFNs, IL-2, -4, -7, -9, -15, and mature T and B cells. The data suggest that FL increases the number of DCs in neonates and possibly in other immune-compromised individuals, which in turn improves IFN-α/β– and IL-12–associated immune responses.

The DNA of an IPV strain of bovid herpesvirus 1 in sacral ganglia during latency after intravaginal infection

Two calves were inoculated intravaginally with a strain of bovid herpesvirus type 1 (BHV-1, IBR/IPV) isolated from a cow with infectious pustular vulvovaginitis (IPV). The animals were killed during a latent stage of infection as characterized by seroconversion, absence of virus shedding and recrudescence of virus shedding after dexamethasone treatment. IPV-virus DNA was detected in 9 out of 20 sacral ganglia of the 2 calves. Of the sections, 7.2% (n = 250) contained 1 cell with IPV-virus DNA, which was restricted to the nucleus of neurons. In agreement with findings on herpes simplex virus infections, the viral DNA of BHV-1 is harbored in the local sensory ganglia. Virological and serological implications of the latent IPV infection are discussed.