Effectiveness of systematic foot and mouth disease mass vaccination campaigns in Argentina.

The objective of this paper is to evaluate the effectiveness of systematic mass vaccination campaigns against foot and mouth disease in Argentina. The analysis was based on an estimation of the proportion of protected animals and protected farms in vaccinated populations, as reflected by levels of antibodies measured in liquid-phase enzyme-linked immunosorbent assay. The analysis was carried out in 49 animal health districts in Buenos Aires province, using data collected from four cross-sectional studies, in 2004, 2007, 2008 and 2011. Cattle were assigned to one of two categories on the basis of correlation between serological titres and expected percentage protection: non-adequately protected (expected protection < 75%) and adequately protected (expected protection ≥ 75%). The proportions of adequately protected cattle and significantly non-adequately protected farms were estimated and compared among sampled locations. Protection was variable among the districts; cattle aged one to two years showed higher levels of protection than cattle six to 12 months old, and the proportion of protected cattle was higher in the more recent studies. The results of the analysis will allow the national animal health service to investigate in depth those districts where protection was lower than the regional background protection. The authors propose that this methodology could be used to evaluate the effectiveness of vaccination campaigns in other countries or zones where systematic foot and mouth disease mass vaccination campaigns are undertaken.

Antigenic and immunogenic spectrum of foot-and-mouth disease vaccine strain O1 Campos against representative viruses of topotypes that circulated in Asia over the past decade.

Identifying vaccine strains to control outbreaks of foot-and-mouth disease virus that could spread to new regions is essential for contingency plans. This is the first report on the antigenic/immunogenic relationships of the South American O1/Campos vaccine strain with representative isolates of the three currently active Asian type O topotypes. Virus neutralization tests using O1/Campos post-vaccination sera derived from cattle and pigs predicted for both species acceptable cross-protection, even after single vaccination, established by r1 values and by expectancy of protection using monovalent or polyvalent vaccines. The results indicate that effective oil vaccines containing the O1/Campos strain can be used against Asian isolates, expanding the scope of O1/Campos strain included in vaccine banks to control emergencies caused by Asian viruses, even on single-dose vaccination, and to cover the need of effective vaccines in Asia during systematic vaccination.

Incidence of group A and atypical rotaviruses in Brazilian pig herds.

The incidence of rotaviruses as a gastroenteritis causal agent in piglets was studied in 19 pig herds of Sao Paulo State, Brazil, during 1985. From 302 diarrhoea samples collected during January (summer), 65 were positive for rotavirus when analysed by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE). Sixty-two of these samples belonged to the classical group A rotavirus, three to atypical rotaviruses (ELISA negative and probably group B) and one elicited a mixed electropherotype of group A and atypical rotavirus and was ELISA positive. Atypical viruses appear to be very fragile and were rapidly degraded upon storage of samples at -20 degrees C. Three herds where atypical rotaviruses were present in January were sampled again in August (winter). Nine atypical isolates out of a total 21 positive samples (assayed by electron microscopy and PAGE) were detected again in two of them.

Incidence of rotavirus in beef herds in Argentina.

Faecal samples were collected from 177 diarrhoeic and 40 healthy calves from 19 farms in Buenos Aires State, Argentina during 1984 and 1985. Samples were examined for rotavirus by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE) of their genomic RNA segments. Rotavirus was found in 95 samples of diarrhoeic calves (53 per cent) and in three healthy calves (7 per cent). All positive samples were tentatively classified as group A on the basis of electropherotype and ELISA test reactivity and exhibited 18 different genomic electropherotypic patterns.

Atypical rotavirus in chickens in Argentina.

In Argentina the presence of rotavirus was investigated in a chicken flock experiencing periodic episodes of diarrhoea during the winter of 1986. All the samples analysed were negative by the enzyme-linked immunosorbent assay (ELISA). However, when samples were observed by electron microscopy, particles which were indistinguishable from standard rotaviruses were detected in some samples. Ten of the 36 samples were positive after polyacrylamide gel electrophoresis (PAGE) analysis, all of them showing the same electropherotype. Based on these results these viruses were classified as rotavirus-like or atypical rotaviruses.

Microbiology of diarrhoea in young beef and dairy calves in Argentina.

Rotavirus, Cryptosporidium sp, and Salmonella spp. were investigated in the faeces of 452 diarrhoeic calves from 36 beef and 33 dairy herds. Animals surveyed were from a few days of age up to approximately 1 month of life. Enterotoxigenic Escherichia coli (ETEC) was studied in 212 calves, aged 15 days or less. The animals were from the Provinces of Buenos Aires (59% of the calves), Córdoba (18%), Santa Fe (16%), Entre Ríos (5%) and La Pampa (2%). A minimum of 4 calves were sampled on each farm. In beef calves rotavirus was excreted by 45.1% of the animals. Cryptosporidium by 30.5% and Sàlmonella serovars Arechabaleta, Livingstone, Panama and Typhimurium by 1.9%. In dairy calves Cryptosporidium was excreted by 29.6%, rotavirus by 23% and Salmonella serovar Dublin by 1.6%, ETEC was not detected in any calf. Rotavirus was the most widespread agent, detected in 32 (88.9%) beef herds and excreted by more than 50% of the calves in half of these herds. In contrast, rotavirus was only detected in 19 (57.5%) dairy herds and was excreted by more than 50% of the calves in 6 of these herds. Crytosporidium oocysts were identified in 27 (75%) beef and in 23 (69.7%) dairy farms. Salmonellosis due to serovar Dublin was associated with diarrhoea in 2 dairy herds. Concurrent infection with two or three agents occurred in 36 (8%) calves and 38 (55.1%) farms; the combination rotavirus-Cryptosporidium was found in 32 (6.9%) calves an in 33 (47.8) farms.

Foot-and-mouth disease vaccine potency testing in cattle using homologous and heterologous challenge strains: precision of the "Protection against Podal Generalisation" test.

The level of protection conferred by foot-and-mouth disease (FMD) vaccines in primovaccinated animals primarily depends on the potency of the vaccine and the relatedness of the vaccine strain and circulating field isolate. The "Gold Standard" FMD vaccine potency test is the in vivo test performed in the target species. The objective of the study was to determine the precision of the in vivo "Protection against Podal Generalisation" (PPG) FMD vaccine potency test in cattle using homologous (vaccine quality control) and heterologous (vaccine matching) viral challenge. The overall level of protection induced by the A(24) Cruzeiro/Brazil/55 vaccine used in six homologous PPG tests was 88.5%. Vaccine accordance (VACC) and vaccine concordance (VCON) were estimated to be 75.9% and 73.7%, respectively. In four heterologous challenge PPG tests, the overall level of cross-protection induced by the A(24) Cruzeiro/Brazil/55 vaccine against A Argentina/2001 challenge was 26.6%, with VACC and VCON values of 65.7% and 59.2%, respectively. Results indicate that the homologous PPG test is more reliable than the European Pharmacopoeia potency test, but that a larger number of animals should be used in order to increase the test's statistical power. In this regard, indirect alternative tests for vaccine potency and vaccine matching merit consideration.

Sequence of a rotavirus gene 4 associated with unique biologic properties.

The genome segment 4 of the simian rotavirus variant SA 11-4F was sequenced. This gene is of probable bovine origin, and it contains a few amino acid differences when compared with other SA 11 variants (4 fm and fem) that were isolated independently and that have fast migration patterns of their gene 4 segments. Hypotheses for the role of sequence changes are made relative to the unique properties of the SA 11-4F variant.

Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity.

We have examined the possible function(s) of the protein VP3 encoded by the rotavirus SA11 genomic segment 3. Viral-associated VP3 in double-shelled and single-shelled particles was shown to bind GTP covalently and reversibly. These properties are similar to the unique characteristics of eukaryotic and viral guanylyltransferases, suggesting that VP3 is associated with a capping enzyme activity. Previous studies have shown that intact viral particles are required for transcription, making it difficult to unequivocally identify the functions of individual proteins within such particles. Characterization of VP3 produced in the baculovirus expression system showed that the expressed VP3 covalently bound GTP. These studies suggest that VP3 alone is the guanylyltransferase. GTP binding also was seen in core virus-like particles and single-shelled virus-like particles that lacked viral nucleic acid and were assembled in insect cells.

Alterations in the sequence of the gene 4 from a human rotavirus after multiple passages in HepG2 liver cells.

The human Wa strain of rotaviruses, initially unable to grow in liver cells, was adapted by multiple passages to grow in HepG2 cells. The genome segment 4 of both the parental and passaged strains was cloned and sequenced. Five amino acid differences (residues 38, 120, 421, 525, and 618) were found in the HepG2-passaged variant compared to the parental Wa strain. Our results support the hypothesis that viral variants that have improved capabilities for infecting liver cells can be generated during infection.

A subviral particle binding domain on the rotavirus nonstructural glycoprotein NS28.

The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.

Monoclonal antibodies to the VP6 of porcine subgroup I rotaviruses reactive with subgroup I and non-subgroup I non-subgroup II strains.

A panel of 10 monoclonal antibodies produced after immunization with two porcine subgroup I rotavirus strains (OSU and A46), and directed against the major inner capsid protein (VP6), fell into six patterns of reactivity when tested against a collection of human and animal group A rotavirus strains. Monoclonal antibodies of pattern I recognized all rotavirus strains. Antibodies of patterns 2 and 3 recognized all subgroup II strains and some, but not all, subgroup I strains. Pattern 4 antibodies identified all subgroup I strains and two strains (H2, equine; CC117, porcine) not reactive with reference subgroup monoclonal antibodies (strains non-I non-II). Pattern 5 antibody exhibited the same reactivity as pattern 4 except for not recognizing the non-I non-II equine strain. Pattern 6 antibodies reacted exclusively with subgroup I and non-I non-II rotaviruses of porcine origin. By competitive binding assays, monoclonal antibodies of patterns 4, 5 and 6 appeared to recognize a single antigenic site, which included at least three overlapping epitopes. In immunoblots all monoclonal antibodies, except one, recognized only the trimeric, but not the monomeric form of VP6.

Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34.

Intermolecular interactions between polypeptide chains often play essential roles in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly. We have initiated studies of the functions of the rotavirus SA114F gene 7 product by sequence analysis and expression in insect cells. This nonstructural protein, NS34, is a slightly acidic protein, and its secondary structure is predicted to be 78% alpha-helix, with several heptad repeats of hydrophobic amino acids being present in its carboxy half. NS34 was found in oligomers when analyzed in insect cells, in SA11-infected MA104 cells, and in cell-free translation reactions. Investigation of the multiple electrophoretically distinct forms of NS34 showed they were all composed of homooligomers. Deletion mutants constructed and tested for oligomerization showed that the carboxy terminus of the protein, containing the predicted heptad repeats, was responsible for oligomerization. A basic region present in NS34 of group A rotaviruses, found to be 40% conserved in NS34 of group C rotavirus, is a candidate for a functional domain of this protein. NS34, which was found to be associated with the cytoskeleton fraction of cells, also interacts with viral RNA. These results make it likely that NS34 plays a central role in the replication and assembly of genomic RNA structures.

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.

Poliovirus vectors are being studied as potential vaccine delivery systems, with foreign genetic sequences incorporated as part of the viral genome. The foreign sequences are expressed as part of the viral polyprotein. Addition of proteolytic cleavage sites at the junction of the foreign polypeptide and the viral proteins results in cleavage during polyprotein processing. The ability of foot-and-mouth disease virus (FMDV) 2A to mediate proteolytic cleavage in the context of poliovirus vectors was studied. The results demonstrate that FMDV 2A is able to generate cleavage of the foreign antigen from the viral polyprotein. A second cleavage event between the FMDV 2A peptide and the foreign protein was also observed.

Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism.

The structures of the rearranged genomic segment 11 of two spontaneous swine rotavirus strains were determined. We found that the rearrangements involved the duplication of normal segment 11 in a head-to-tail orientation, and partial deletions in both monomers. The open reading frame for VP11, the protein encoded by normal segment 11, was maintained. We also showed that the two rearranged genes were transcribed into RNA molecules of the same length as their corresponding genomic segments.

Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.

Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.

Rearrangement of genomic segment 11 in two swine rotavirus strains.

We have recently reported the isolation of two group A swine rotaviruses each lacking normal genomic RNA segment 11 and showing instead one extra segment that migrated abnormally on gel electrophoresis. Hybridization studies performed with segment-specific probes and with a purified abnormal RNA segment showed that the extra bands had sequence homology to normal segment 11. Analysis of protein profiles of normal and rearranged strains showed that the gene product of segment 11 had no apparent change in its relative electrophoretic migration, suggesting that the rearranged genes remained functional.

A bovine rotavirus serotype 1: serologic characterization of the virus and nucleotide sequence determination of the structural glycoprotein VP7 gene.

Bovine rotavirus T449 was isolated from feces of a calf with diarrhea. Serological characterization by serotype-specific monoclonal antibodies showed that the T449 virus belonged to serotype 1. This is the first report of a bovine rotavirus that does not belong to serotype 6, 8, or 10. The serotype 1 designation was confirmed by using an immunoperoxidase focus neutralization assay. The gene encoding the major neutralization antigen (VP7) was cloned and its nucleotide sequence was determined. The sequence obtained was 1062 bp in length and contained an open reading frame corresponding to 326 amino acid residues. Comparative analysis of the deduced amino acid sequence with the corresponding sequence of the human serotype 1 rotavirus strain, Wa, revealed a 90% identity. When compared to the predicted amino acid sequence of VP7 protein of the other serotypes an overall divergence of 20 to 25% was detected. These data show that the serological typing agrees with the result of the genetic analysis.

Attenuated poliovirus strain as a live vector: expression of regions of rotavirus outer capsid protein VP7 by using recombinant Sabin 3 viruses.

The ability to express heterologous antigens from attenuated poliovirus strains suggests the potential for use as live vectored vaccines. Full- or partial-length sequences of the gene encoding rotavirus major outer capsid protein VP7 were cloned into the open reading frame of a full-length cDNA copy of poliovirus Sabin type 3. They were inserted either at the 5' end or immediately after the capsid protein coding region, at the junction between precursors P1 and P2. A protease cleavage site for 3C protease was introduced 3' to the foreign sequences to enable proteolytic processing of the antigen from the poliovirus polyprotein. Infectious viruses were generated from several of the DNA constructs, and the presence of the foreign gene sequences was confirmed by reverse transcription of the viral RNA and PCR amplification. Viruses with inserts of about 300 bases maintained the foreign sequences during passage in Vero cells. Viruses carrying larger sequences were unstable, and deletions were generated within the foreign sequences. Expression of the VP7 polypeptides was demonstrated by immunoprecipitation with specific antiserum of labeled proteins from cells infected with Sabin 3 recombinant viruses. Comparative studies of RNA synthesis showed similar kinetics for Sabin 3 and the Sabin 3/VP7 recombinants. One-step growth curves showed that production of recombinant viruses was slower than that of Sabin 3 and that the final titers were 1 to 1.5 logs lower. Accumulation of VP7-containing precursors in infected cells suggests that slow cleavage at the engineered 3C protease site may be a limiting step in the growth of these recombinant Sabin polioviruses and may influence the permissible size of foreign sequence to be inserted.