Steven J. Pauszek

The region between the two polyprotein initiation codons of foot-and-mouth disease virus is critical for virulence in cattle.

To explore the role in viral pathogenesis of the region located between the two functional AUG (inter-AUG) in foot-and-mouth disease virus (FMDV), we derived viruses containing transposon (tn) inserts from a mutagenized cDNA infectious clone of FMDV (pA24-WT). Mutant viruses containing an in-frame 57-nt transposon insertion grew at a slower rate and had a smaller plaque size phenotype than the parental virus (A24-WT). A mutant virus containing a 51-nt deletion in inter-AUG had a similar phenotype in cell culture to that of A24-WT. When tested by aerosol inoculation in cattle (3 animals per virus), the deletion mutant was fully virulent as was A24-WT. Mutant viruses containing insertions in inter-AUG did not cause clinical disease or viremia. However, viruses that partially or totally removed the tn insertion during animal infection reverted to virulence in 2 inoculated steers. Therefore, this study identified inter-AUG as an FMDV viral virulence determinant in cattle infected by aerosol route.

Vesicular Stomatitis Virus Glycoprotein Is a Determinant of Pathogenesis in Swine, a Natural Host

ABSTRACT There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-GI); (ii) two copies of the G gene, one from each serotype (VSIV-GNJGI); or (iii) a single copy of the GNJ gene instead of the GI gene (VSIV-GNJ). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-GI), VSNJV (VSIV-GNJ), or both (VSIV-GNJGI), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-GNJ and VSIV-GNJGI were attenuated. However, in swine, a natural host for VSV, the GNJ glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-GI. These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

ABSTRACT Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvβ6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle. IMPORTANCE The objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus strains occurring in Mexico and North and Central America. A comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America was tested by the real-time RT-PCR assay and virus isolation. Overall, clinical sensitivity and specificity of the real-time RT-PCR were 83% and 99%, respectively. Interestingly, VS virus isolates originating from a specific region of Costa Rica were not detected by real-time RT-PCR. Sequence comparisons of these viruses with the real-time RT-PCR probe and primers showed mismatches in the probe and forward and reverse primer regions. Additional lineage-specific primers and a probe corrected the lack of detection of the missing genetic lineage. Thus, this assay reliably identified existing Mexican and Central American VS viruses and proved readily adaptable as new VS viruses were encountered. An important secondary result of this research was the collection of hundreds of new VS virus isolates that provide a foundation from which many additional studies can arise.

Foot-and-mouth disease virus virulence in cattle is co-determined by viral replication dynamics and route of infection.

Early events in the pathogenesis of foot-and-mouth disease virus (FMDV) infection in cattle were investigated through aerosol and intraepithelial lingual (IEL) inoculations of a cDNA-derived FMDV-A24 wild type virus (FMDV-WT) or a mutant derived from the same clone (FMDV-Mut). After aerosolization of FMDV-WT, primary infection sites had significantly greater quantities of FMDV, viral RNA, and type I/III interferon (IFN) activity compared to corresponding tissues from cattle infected with FMDV-Mut. Additionally, FMDV-WT-infected cattle had marked induction of systemic IFN activity in serum. In contrast, FMDV-Mut aerosol-infected cattle did not manifest systemic IFN response nor had viremia. Interestingly, IEL inoculation of FMDV-Mut in cattle restored the virulent phenotype and systemic IFN response. These data indicate that the attenuated phenotype in cattle is associated with decreased replicative efficiency, reflected by decreased innate response. However, attenuation is abrogated by bypassing the common primary infection sites, inducing accelerated viral replication at the inoculation site.

Characterization of the full-length genomic sequences of vesicular stomatitis Cocal and Alagoas viruses

In Brazil and Argentina, vesicular stomatitis (VS) is caused by distinct viral strains serologically related to the classical vesicular stomatitis virus Indiana (VSIV), namely VS Indiana-2 (VSIV-2) and VS Indiana-3 (VSIV-3). Here we describe the full-length genomic sequences and organization of the prototype strains of VSIV-2 Cocal virus (COCV) and VSIV-3 Alagoas virus (VSAV). These viruses showed similar genomic organizations to VSIV field isolates except that the non-structural C′/C proteins, markedly conserved throughout the vesiculoviruses, were absent in VSAV. Phylogenetic analyses consistently grouped COCV, VSAV and VSIV in a monophyletic group distinct from VSNJV, supporting the classification of these viruses within the Indiana serogroup.

Spatial and phylogenetic analysis of vesicular stomatitis virus over-wintering in the United States.

From 2004 through 2006, 751 vesicular stomatitis (VS) outbreaks caused by vesicular stomatitis virus serotype New Jersey (VSNJV) were reported in nine states of the southwestern United States. The normal model of the spatial scan statistic and phylogenetic techniques were used to assess whether the spatial and genetic relations among VSNJV outbreaks were consistent with the hypothesis that VSNJV over-wintered in specific regions of the southwestern United States infected in 2004 and 2005, respectively. Use of the spatial scan statistic led to the identification of two clusters of outbreaks for which the Euclidean distance to the nearest outbreak reported in the previous or following year, whichever was shorter, was significantly (P<0.01) shorter than the epidemics (2004-2006) mean. Clusters were centered at Colorado and Wyoming and included 375 and 21 outbreaks, respectively. Results were supported by the phylogenetic analysis of 49 VSV samples collected from 2004 through 2006 in the United States and 10 VSV samples originated from Mexico. These findings, which were displayed using a publicly accessible web-based system referred to as the FMD BioPortal, were consistent with over-wintering of specific sub-lineages of VSNJV in a limited geographical region of the United States affected by a VS epidemic in 2005 and 2006.

Serotype Diversity of Foot‐and‐Mouth‐Disease Virus in Livestock without History of Vaccination in the Far North Region of Cameroon

Little information is available about the natural cycle of foot-and-mouth disease (FMD) in the absence of control measures such as vaccination. Cameroon presents a unique opportunity for epidemiological studies because FMD vaccination is not practiced. We carried out a prospective study including serological, antigenic and genetic aspects of FMD virus (FMDV) infections among different livestock production systems in the Far North of Cameroon to gain insight into the natural ecology of the virus. We found serological evidence of FMDV infection in over 75% of the animals sampled with no significant differences of prevalence observed among the sampled groups (i.e. market, sedentary, transboundary trade and mobile). We also found antibodies reactive to five of the seven FMDV serotypes (A, O, SAT1, SAT2 and SAT3) among the animals sampled. Finally, we were able to genetically characterize viruses obtained from clinical and subclinical FMD infections in Cameroon. Serotype O viruses grouped into two topotypes (West and East Africa). SAT2 viruses grouped with viruses from Central and Northern Africa, notably within the sublineage causing the large epidemic in Northern Africa in 2012, suggesting a common origin for these viruses. This research will guide future interventions for the control of FMD such as improved diagnostics, guidance for vaccine formulation and epidemiological understanding in support of the progressive control of FMD in Cameroon.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses.

This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Growth and molecular evolution of vesicular stomatitis serotype New Jersey in cells derived from its natural insect–host: evidence for natural adaptation

In this study we evaluated the growth and molecular evolution of a natural isolate of VSV-NJ (89GAS) from sand flies in cells derived from sand flies (LL5), mosquitoes (C6/36) or hamsters (BHK21). Two VSV-NJ natural isolates of mammalian origin were used for comparison. For each virus we measured: (i) ability to grow in one-step growth curve or after serial passage on each cell type; (ii) ability to induce persistent infection, and (iii) genetic stability of the glycoprotein gene (G) after serial passage in each cell type. Sand fly virus 89GAS grew to higher titers in insect cells compared with viruses of mammalian origin and showed increasing titers with each passage only in C6/36 cells. All viruses established productive persistent infections in both mosquito and sand fly cells but only LL5 cells yielded sustained high virus titers for periods of up to 81 days. Analyses of the consensus sequences of the G gene from each virus after 0, 10 or up to 25 passages in each cell line showed nucleotide substitution rates between 1.39 x l0(-4) and 6.95 x l0(-5). The majority of these changes were non-synonymous, suggesting positive selection. We did not detect increased nucleotide substitution rates on the G gene of 89GAS after passage in cell lines of mosquito or mammalian origin, nor in viruses of mammalian origin after passage in insect cells. This indicates that although VSV G is positively selected in vitro by the insect cell environment, this does not fully explain VSV natural adaptation. This is the first evidence of naturally occurring adaptation of VSV to cells derived from its natural host.