Hybrid Frequencies Confirm Limit to Coinfection in the RNA Bacteriophage φ6

Coinfection of the same host cell by multiple viruses may lead to increased competition for limited cellular resources, thus reducing the fitness of an individual virus. Selection should favor viruses that can limit or prevent coinfection, and it is not surprising that many viruses have evolved mechanisms to do so. Here we explore whether coinfection is limited in the RNA bacteriophage 6 that infects Pseudomonas phaseolicola. We estimated the limit to coinfection in 6 by comparing the frequency of hybrids produced by two marked phage strains to that predicted by a mathematical model based on differing limits to coinfection. Our results provide an alternative method for estimating the limit to coinfection and confirm a previous estimate between two to three phages per host cell. In addition, our data reveal that the rate of coinfection at low phage densities may exceed that expected through random Poisson sampling. We discuss whether phage 6 has evolved an optimal limit that balances the costly and beneficial fitness effects associated with multiple infections.     

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ?-propionolactone (?-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.     

Analysis of Subcellular Localization and Pathogenicity of <i>Plum Bark Necrosis Stem-Pitting Associated Virus</i> Protein P6

Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many Prunus species in several countries, causing significant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101. Infiltration of Nicotiana benthamiana (N. benthamiana) with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, infiltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no significant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms, and the mode of action of PBNSPaV-P6 protein remains to be further studied.     

Important factors for testing barrier materials with surrogate viruses.

This study evaluated bacteriophages phi X174, T7, PRD1, and phi 6 as possible surrogates for pathogenic human viruses to challenge barrier materials and demonstrated some important factors for their use. Chemical incompatibility with test material was demonstrated when lipid-enveloped phi 6 was inactivated by an aqueous eluate of vinyl gloves, but 0.5% calf serum protected phi 6 from the eluate. Low concentrations (2%) of calf serum also prevented the exaggerated binding of the bacteriophages to filters. Recovery of viruses from surfaces decreased with increasing time before recovery. Penetration through punctures displayed different types of kinetics. The combined data indicate that (i) some bacteriophages may serve as surrogate viruses, (ii) experimental conditions determine whether a particular virus is appropriate as a challenge, and (iii) phi X174 is an excellent choice as a surrogate virus to test barrier materials. The data further indicate that before barrier materials are challenged with viruses, adequate tests should be performed to ensure that the virus is compatible with the test material and test conditions, so that meaningful data will result.     

Important factors for testing barrier materials with surrogate viruses.

This study evaluated bacteriophages phi X174, T7, PRD1, and phi 6 as possible surrogates for pathogenic human viruses to challenge barrier materials and demonstrated some important factors for their use. Chemical incompatibility with test material was demonstrated when lipid-enveloped phi 6 was inactivated by an aqueous eluate of vinyl gloves, but 0.5% calf serum protected phi 6 from the eluate. Low concentrations (2%) of calf serum also prevented the exaggerated binding of the bacteriophages to filters. Recovery of viruses from surfaces decreased with increasing time before recovery. Penetration through punctures displayed different types of kinetics. The combined data indicate that (i) some bacteriophages may serve as surrogate viruses, (ii) experimental conditions determine whether a particular virus is appropriate as a challenge, and (iii) phi X174 is an excellent choice as a surrogate virus to test barrier materials. The data further indicate that before barrier materials are challenged with viruses, adequate tests should be performed to ensure that the virus is compatible with the test material and test conditions, so that meaningful data will result.     

Vector-Borne Transmission Imposes a Severe Bottleneck on an RNA Virus Population

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller''s ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study.     

Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC₅₀ = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.     

Biology and Evolution of Beneficial and Detrimental Viruses of Animals, Plants, and Fungi

Virtually every type of organism may serve as a host for viruses. In some hosts, virus presence may be considered beneficial to humans; in other hosts, viruses are considered detrimental. Examples of viruses that are considered beneficial to humans include those that are used for biological control of organisms that themselves are considered detrimental to humans, such as plant pathogenic fungi. Viruses are extremely variable in terms of morphology, structure, and genome organization. However, viruses that attack hosts from different kingdoms may be related, deriving from the same phylogeny. This paper summarizes some of the properties of three related families of viruses that attack hosts in different kingdoms: the animal-infecting Picornaviridae, the plant-infecting Potyviridae, and the fungus-infecting Hypoviridae. Properties of these viruses that set them apart from each other and factors that may affect their evolution are discussed.     

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

Background  

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.     

Efficient functional pseudotyping of oncoretroviral and lentiviral vectors by Venezuelan equine encephalitis virus envelope proteins

Murine oncoretroviruses and lentiviruses pseudotyped with envelope proteins of alphaviruses have shown great potential in providing broad-host-range, stable vectors for gene therapy. Unlike vesicular stomatitis virus G protein-pseudotyped vectors, they are not neutralized by complement and do not appear to cause significant tissue damage. Here we report the production of murine oncoretroviral and lentiviral vectors pseudotyped with the envelope proteins of Venezuelan equine encephalitis virus (VEEV). When optimized, these pseudotypes achieve titers of 10⁶ CFU/ml, which is 5- to 10-fold higher than for previous vectors pseudotyped with envelope proteins from other alphaviruses. They can also be concentrated or stored frozen without significant loss of infectivity. Consistent with the tropism of the envelope donor, they transduce a broad array of human cell types, including lung epithelial cells, neuronal cells, lymphocytes, and fibroblasts. Infection is blocked by agents that inhibit endosomal acidification and by neutralizing antibodies against VEEV. These observations indicate that the pseudotypes present native epitopes on their surface and enter through a VEEV envelope-dependent, pH-sensitive mechanism. The fact that the pseudotypes are unaffected by sera reactive to other alphaviruses indicates that they may be useful when successive gene therapies are required in the presence of an active immune response. In this case, having an array of alphavirus-based vectors with similar cell tropisms would be highly advantageous. These vectors may also be useful in diagnostic assays in which infectious VEEV is undesirable but immune reactivity to native epitopes is required.     

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.     

Seasonal dynamics and co‐occurrence patterns of honey bee pathogens revealed by high‐throughput RT‐qPCR analysis

The health of the honey bee Apis mellifera is challenged by introduced parasites that interact with its inherent pathogens and cause elevated rates of colony losses. To elucidate co‐occurrence, population dynamics, and synergistic interactions of honey bee pathogens, we established an array of diagnostic assays for a high‐throughput qPCR platform. Assuming that interaction of pathogens requires co‐occurrence within the same individual, single worker bees were analyzed instead of collective samples. Eleven viruses, four parasites, and three pathogenic bacteria were quantified in more than one thousand single bees sampled from sixteen disease‐free apiaries in Southwest Germany. The most abundant viruses were black queen cell virus (84%), Lake Sinai virus 1 (42%), and deformed wing virus B (35%). Forager bees from asymptomatic colonies were infected with two different viruses in average, and simultaneous infection with four to six viruses was common (14%). Also, the intestinal parasites Nosema ceranae (96%) and Crithidia mellificae/Lotmaria passim (52%) occurred very frequently. These results indicate that low‐level infections in honey bees are more common than previously assumed. All viruses showed seasonal variation, while N. ceranae did not. The foulbrood bacteria Paenibacillus larvae and Melissococcus plutonius were regionally distributed. Spearman's correlations and multiple regression analysis indicated possible synergistic interactions between the common pathogens, particularly for black queen cell virus. Beyond its suitability for further studies on honeybees, this targeted approach may be, due to its precision, capacity, and flexibility, a viable alternative to more expensive, sequencing‐based approaches in nonmodel systems.     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.     

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model''s predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.     

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.     

Eastern and Venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-alpha/beta) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-alpha/beta induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

Potential sources of the 1995 Venezuelan equine encephalitis subtype IC epidemic

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962-1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962-1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.