Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus

Low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate into highly pathogenic (HPAI) variants, causing high mortality in poultry. The switch to high pathogenicity is poorly understood, and evidence from the field is scarce. This study provides direct evidence for LPAI to HPAI mutation from a turkey farm during an H7N3 outbreak in the Netherlands. At the farm, only mild clinical symptoms were reported, but the intravenous pathogenicity index measured for the virus isolated from the infected turkeys was consistent with a highly pathogenic virus. Using deep-sequencing, we showed that a minority of HPAI virus (0.06%) was present in the virus preparation. Analysis of different organs of the infected turkeys showed the highest percentage of HPAI virus was present in the lung (4.4%). The HPAI virus contained a 12-nucleotide insertion in the hemagglutinin (HA) cleavage site that was introduced by a single event, as no intermediates with shorter inserts were identified. The HPAI virus was rapidly selected in chickens, after both intravenous and intranasal/intratracheal inoculation with the mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence of HPAI viruses. This knowledge may be used for the timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field.

DEVELOPMENT OF A TEST-SYSTEMS FOR THE IDENTIFICATION OF THE SOIL BORNED TOBACCO RATTLE VIRUS BASED ON THE USE OF ELISA

On the base of the isolate TRV PV-0361 of the tobacco rattle virus from the commercial collection of the DSMZ company (Germany) have been developed methods for maintaining the virus in vitro culture on N. clevelandii plants, propagation the virus in the spring-summer period in greenhouse conditions, “soft” isolation of a purified virus preparation by clarifying leaf juice with low-speed centrifugation and treatment with non-ionic detergent Triton-X-100 followed by precipitation of the virus with polyethylene glycol-6000 and three-fold ultracentrifugation using sucrose cushion, sucrose concentration gradient and differential centrifugation. The purified virus preparation was used for producing rabbit antiserums according to the scheme we worked out. The obtained antiserum had the following titers - specific 1: 5 · 105, non-specific 1: 8 · 103. Based on antibodies isolated from this antiserum, coating antibodies and peroxidase conjugates were obtained, which made it possible to create ELISA test systems for determining TRV with sensitivity of about 12-16 ng/ml. The resulting test systems can be used in practical work on quality control and certification of seed potatoes.

Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.

Ion-Exchange Membrane Chromatography as an Alternative Method of Separation of Potato y Virus

Procedures of separation of virus particles from a plant material are multistage. Furthermore often they are difficult in terms of methodology and require use of expensive, highly specialist equipment and yield of separation is often low. The antigen obtained is often degraded and contains admixtures of other proteins. Therefore, generation of high quality and specificity antibodies based on such antigen is very difficult and quality of the antibodies has impact on reliability, sensitivity and unambiguity of results of immunodiagnostic tests (e.g. ELISA) that are currently conventionally used to detect vegetable viruses. In this study three conventionally-performed methods of separation of potato virus Y (PVY) were compared and a method of separation based on membrane chromatography, as an alternative separation technique, has been presented. It has been demonstrated that in proper process conditions good quality virus preparation can be obtained.