Efficacy of multivalent recombinant herpesvirus of turkey vaccines against high pathogenicity avian influenza, infectious bursal disease, and Newcastle disease viruses.

Vaccines are an essential tool for the control of viral infections in domestic animals. We generated recombinant vector herpesvirus of turkeys (vHVT) vaccines expressing computationally optimized broadly reactive antigen (COBRA) H5 of avian influenza virus (AIV) alone (vHVT-AI) or in combination with virus protein 2 (VP2) of infectious bursal disease virus (IBDV) (vHVT-IBD-AI) or fusion (F) protein of Newcastle disease virus (NDV) (vHVT-ND-AI). In vaccinated chickens, all three vHVT vaccines provided 90-100% clinical protection against three divergent clades of high pathogenicity avian influenza viruses (HPAIVs), and significantly decreased number of birds and oral viral shedding titers at 2 days post-challenge compared to shams. Four weeks after vaccination, most vaccinated birds had H5 hemagglutination inhibition antibody titers, which significantly increased post-challenge. The vHVT-IBD-AI and vHVT-ND-AI vaccines provided 100% clinical protection against IBDVs and NDV, respectively. Our findings demonstrate that multivalent HVT vector vaccines were efficacious for simultaneous control of HPAIV and other viral infections.

Efficacy of recombinant Marek's disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses.

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.

Cross-Protection by Inactivated H5 Prepandemic Vaccine Seed Strains against Diverse Goose/Guangdong Lineage H5N1 Highly Pathogenic Avian Influenza Viruses.

The highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 lineage (Gs/GD) is endemic in poultry across several countries in the world and has caused sporadic lethal infections in humans. Vaccines are important in HPAIV control both for poultry and in prepandemic preparedness for humans. This study assessed inactivated prepandemic vaccine strains in a One Health framework across human and agricultural and wildlife animal health, focusing on the genetic and antigenic diversity of field H5N1 Gs/GD viruses from the agricultural sector and assessing cross-protection in a chicken challenge model. Nearly half (47.92%) of the 48 combinations of vaccine and challenge viruses examined had bird protection of 80% or above. Most vaccinated groups had prolonged mean death times (MDT), and the virus-shedding titers were significantly lower than those of the sham-vaccinated group (P ≤ 0.05). The antibody titers in the prechallenge sera were not predictive of protection. Although vaccinated birds had higher titers of hemagglutination-inhibiting (HI) antibodies against the homologous vaccine antigen, most of them also had lower or no antibody titer against the challenge antigen. The comparison of all parameters and homologous or closely related vaccine and challenge viruses gave the best prediction of protection. Through additional analysis, we identified a pattern of epitope substitutions in the hemagglutinin (HA) of each challenge virus that impacted protection, regardless of the vaccine used. These changes were situated in the antigenic sites and/or reported epitopes associated with virus escape from antibody neutralization. As a result, this study highlights virus diversity, immune response complexity, and the importance of strain selection for vaccine development to control H5N1 HPAIV in the agricultural sector and for human prepandemic preparedness. We suggest that the engineering of specific antigenic sites can improve the immunogenicity of H5 vaccines.IMPORTANCE The sustained circulation of highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 (Gs/GD) lineage in the agricultural sector and some wild birds has led to the evolution and selection of distinct viral lineages involved in escape from vaccine protection. Our results using inactivated vaccine candidates from the human pandemic preparedness program in a chicken challenge model identified critical antigenic conformational epitopes on H5 hemagglutinin (HA) from different clades that were associated with antibody recognition and escape. Even though other investigators have reported epitope mapping in the H5 HA, much of this information pertains to epitopes reactive to mouse antibodies. Our findings validate changes in antigenic epitopes of HA associated with virus escape from antibody neutralization in chickens, which has direct relevance to field protection and virus evolution. Therefore, knowledge of these immunodominant regions is essential to proactively develop diagnostic tests, improve surveillance platforms to monitor AIV outbreaks, and design more efficient and broad-spectrum agricultural and human prepandemic vaccines.

Protection of White Leghorn chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses.

Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus.

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.

A computationally designed H5 antigen shows immunological breadth of coverage and protects against drifting avian strains.

Since the first identification of the H5N1 Goose/Guangdong lineage in 1996, this highly pathogenic avian influenza virus has spread worldwide, becoming endemic in domestic poultry. Sporadic transmission to humans has raised concerns of a potential pandemic and underscores the need for a broad cross-protective influenza vaccine. Here, we tested our previously described methodology, termed Computationally Optimized Broadly Reactive Antigen (COBRA), to generate a novel hemagglutinin (HA) gene, termed COBRA-2, that was based on H5 HA sequences from 2005 to 2006. The COBRA-2 HA virus-like particle (VLP) vaccines were used to vaccinate chickens and the immune responses were compared to responses elicited by VLP's expressing HA from A/whooper swan/Mongolia/244/2005 (WS/05), a representative 2005 vaccine virus from clade 2.2. To support this evaluation a hemagglutination inhibition (HAI) breadth panel was developed consisting of phylogenetically and antigenically diverse H5 strains in circulation from 2005 to 2006, as well as recent drift variants (2008 - 2014). We found that the COBRA-2 VLP vaccines elicited robust HAI titers against this entire breadth panel, whereas the VLP vaccine based upon the recommended WS/05 HA only elicited HAI responses against a subset of strains. Furthermore, while all vaccines protected chickens against challenge with the WS/05 virus, only the human COBRA-2 VLP vaccinated birds were protected (80%) against a recent drifted clade 2.3.2.1B, A/duck/Vietnam/NCVD-672/2011 (VN/11) virus. This is the first report to demonstrate seroprotective antibody responses against genetically diverse clades and sub-clades of H5 viruses and protective efficacy against a recent drifted variant using a globular head based design strategy.

Very Virulent Infectious Bursal Disease Virus Produces More-Severe Disease and Lesions in Specific-Pathogen-Free (SPF) Leghorns Than in SPF Broiler Chickens.

Infectious bursal disease virus (IBDV) is an important pathogen of chickens causing negative economic impacts in poultry industries worldwide. IBDV has a variable range of virulence, with very virulent (vvIBDV) strains being responsible for the greatest losses from mortality and decreased performance. Previous vvIBDV studies using conventional broilers reported resistance to lethal effects and decreased performance as compared to specific-pathogen-free (SPF) layers, but the potential contribution of the conventional vs. SPF status to resistance has not been examined. In this study we compared differences in the acute pathologic effects of infection by the California rA strain of vvIBDV for SPF white leghorn egg-laying chickens and SPF white Plymouth Rock broiler chickens over a 7-day experimental period. Based on the clinical signs and mortality observed, as well as on the more-severe pathologic changes in lymphoid tissues and kidneys, white leghorns were shown to be more susceptible to the deleterious effects of vvIBDV infection than were white Plymouth Rocks. This study provides important information on the impact of chicken breed on susceptibility to vvIBDV and the absence of impact from conventional vs. SPF status on the outcome.

Vaccination of chickens decreased Newcastle disease virus contamination in eggs.

Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens.

Variation in protection of four divergent avian influenza virus vaccine seed strains against eight clade 2.2.1 and 2.2.1.1. Egyptian H5N1 high pathogenicity variants in poultry.

BACKGROUND: Highly pathogenic (HP) H5N1 avian influenza virus (AIV) was introduced to Egyptian poultry in 2006 and has since become enzootic. Vaccination has been utilized as a control tool combined with other control methods, but for a variety of reasons, the disease has not been eradicated. In 2007, an antigenically divergent hemagglutinin subclade, 2.2.1.1, emerged from the original clade 2.2.1 viruses. OBJECTIVES: The objective was to evaluate four diverse AIV isolates for use as vaccines in chickens, including two commercial vaccines and two additional contemporary isolates, against challenge with numerous clade 2.2.1 and clade 2.2.1.1 H5N1 HPAIV Egyptian isolates to assess the variation in protection among different vaccine and challenge virus combinations. METHODS: Vaccination-challenge studies with four vaccines and up to eight challenge strains with each vaccine for a total of 25 vaccination-challenge groups were conducted with chickens. An additional eight groups served as sham-vaccinated controls. Mortality, mean death time, morbidity, virus, and pre-challenge antibodies were evaluated as metrics of protection. Hemagglutination inhibition data were used to visualize the antigenic relatedness of the isolates. RESULTS AND CONCLUSIONS: Although all but one vaccine-challenge virus combination significantly reduced shed and mortality as compared to sham vaccinates, there were differences in protection among the vaccines relative to one another based on challenge virus. This emphasizes the difficulty in vaccinating against diverse, evolving virus populations, and the importance of selecting optimal vaccine seed strains for successful HPAIV control.

Genetic diversity of 3' region of glycoprotein D gene of bovine herpesvirus 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3' region of gD gene (gD3') of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3' sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3'. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3' potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3' conservation than previously reported.

Protection against H7N3 high pathogenicity avian influenza in chickens immunized with a recombinant fowlpox and an inactivated avian influenza vaccines.

Beginning on June 2012, an H7N3 highly pathogenic avian influenza (HPAI) epizootic was reported in the State of Jalisco (Mexico), with some 22.4 million chickens that died, were slaughtered on affected farms or were preemptively culled on neighboring farms. In the current study, layer chickens were vaccinated with a recombinant fowlpox virus vaccine containing a low pathogenic AI (LPAI) H7 gene insert (rFPV-H7-AIV) and an inactivated oil-emulsified H7N3 AIV vaccine, and subsequently challenged against the Jalisco H7N3 HPAIV. All vaccine combinations provided similar and significant protection against mortality, morbidity, and shedding of challenge virus from the respiratory and gastrointestinal tracts. Serological data also suggested analogous protection from HPAIV among immunized birds. Control of the recent Jalisco AIV infection could be achieved by using various combinations of the two vaccines tested. Even though a single dose of rFPV-H7-AIV vaccine at 1-day-of-age would be the most pragmatic option, optimal protection may require a second dose of vaccine administered in the field.

Domestic goose as a model for West Nile virus vaccine efficacy.

West Nile virus (WNV) is an emergent pathogen in the Americas, first reported in New York during 1999, and has since spread across the USA, Central and South America causing neurological disease in humans, horses and some bird species, including domestic geese. No WNV vaccines are licensed in the USA for use in geese. This study reports the development of a domestic goose vaccine efficacy model, based on utilizing multiple parameters to determine protection. To test the model, 47 geese were divided in two experiments, testing five different vaccine groups and two sham groups (challenged and unchallenged). Based on the broad range of results for individual metrics between the Challenged-Sham and Unchallenged-Sham groups, the best parameters to measure protection were Clinical Pathogenicity Index (CPI), plasma virus positive geese on days 1-4 post-inoculation and plasma virus titers, and brain histological lesion rates and severity scores. Compared to the Challenged-Sham group, the fowlpox virus vectored vaccine with inserts of WNV prM and E proteins (vFP2000) provided the best protection with significant differences in all five metrics, followed by the canarypox virus vectored vaccine with inserts of WNV prM and E proteins (vCP2018) with four metrics of protection, recombinant vCP2017 with three metrics and WNV E protein with one. These data indicate that domestic geese can be used in an efficacy model for vaccine protection studies using clinical, plasma virological and brain histopathological parameters to evaluate protection against WNV challenge.

Nucleotide sequencing and phylogenetic analysis of the 3' region of glycoprotein C gene of South American bovine herpesviruses 1 and 5.

We herein describe a molecular analysis based on the 3' region of the glycoprotein C gene of 45 bovine herpesvirus (BoHV) isolates from Brazil (n=41), Uruguay (n=2) and Argentina (n=2). Nucleotide (nt) sequencing and alignment of 333 nt revealed levels of similarity ranging from 99.1% to 100% among BoHV-1 sequences (n=12); 96.2-100% among BoHV-5 sequences (n=32); and 77.7-90.3% between BoHV-1 and BoHV-5 sequences. The phylogenetic tree reconstruction provided a clear distinction between BoHV-1 and BoHV-5, and BoHV-1 into subtypes BoHV-1.1 and BoHV-1.2. The isolate SV 453/93 (BoHV-1 associated with genital disease) could not be included within BoHV-1 subtypes since it presented a markedly distinct nt and amino acid (aa) deduced sequences. A transmembrane domain of 24 aa and the putative cytoplasmic tail of 8 aa were identified and mapped. These results indicate that this genome region represents a suitable target for phylogenetic subgrouping of BoHV-1 and BoHV-5 isolates and, perhaps, for understanding evolutionary relationships.

Serum and egg yolk antibody detection in chickens infected with low pathogenicity avian influenza virus.

Surveillance for low pathogenicity avian influenza virus (LPAIV) infections has primarily relied on labor-intensive collection and serological testing of serum, but for many poultry diseases, easier-to-collect yolk samples have replaced serum for surveillance testing. A time-course LPAIV infection study in layers was performed to evaluate the utility of antibody detection in serum vs. egg yolk samples. Layers inoculated with the LPAIV A/Bobwhite Quail/Pennsylvania/20304/98 (H7N2) were tested for antibody levels in the serum and egg yolk by using the agar gel immunodiffusion test (AGID), hemagglutination-inhibition test (HI), and a commercially available enzyme-linked immunosorbent assay (ELISA). Anti-influenza specific antibodies were detected in the serum as early as 7 days postinoculation (DPI), and the majority of the hens remained positive until 42 DPI. Antibodies in the egg yolk were first detected by AGID at 7 DPI, which was also the first day of detection in serum. However, the majority of the eggs were positive by all techniques at 11 DPI and remained positive until 42 DPI, at which time the number of AGID+ and HI+ samples declined slightly as compared to ELISA+ samples. These results suggest that egg yolk can be an alternative to serum for flock serological surveillance against LPAIV infections, and the three methods (AGID, HI, and ELISA) will give similar results for first 42 days after infection, although AGID may give earlier positive response.

Experimental infection with low and high pathogenicity H7N3 Chilean avian influenza viruses in Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera).

Two different wild duck species common in Chile and neighboring countries, Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera), were intranasally inoculated with 10(6) mean embryo infective dose (EID50) of the H7N3 low pathogenicity (LP) avian influenza virus (AIV) (A/chicken/Chile/176822/02) or high pathogenicity (HP) AIV (A/chicken/Chile/ 184240-1/02), in order to study the infectivity and pathobiology of these viruses. None of the virus-inoculated ducks had clinical signs or died, but most seroconverted by 14 days postinoculation (DPI), indicating a productive virus infection. Both LPAIV and HPAIV were isolated from oral swabs from two of six Chiloe wigeons and from oral and/or cloacal swabs from all five of the cinnamon teal at 2 DPI. Both LPAIV and HPAIV were efficiently transmitted to cinnamon teal contacts but not to Chiloe wigeon contacts. This study demonstrates that the cinnamon teal and Chiloe wigeons were susceptible to infection with both Chilean H7N3 LPAIV and HPAIV, but only the cinnamon teal showed contact transmission of the virus between birds, suggesting that the cinnamon teal has the potential to be a reservoir for these viruses, especially the LPAIV, as was demonstrated in 2001 with isolation of a genetically related H7N3 LPAIV strain in a cinnamon teal in Bolivia. However, the definitive source of the H7N3 Chilean LPAIV still remains unknown.

Two small RNAs encoded within the first 1.5 kilobases of the herpes simplex virus type 1 latency-associated transcript can inhibit productive infection and cooperate to inhibit apoptosis.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected trigeminal ganglionic sensory neurons. Expression of the first 1.5 kb of LAT coding sequences is sufficient for the wild-type reactivation phenotype in small animal models of infection. The ability of the first 1.5 kb of LAT coding sequences to inhibit apoptosis is important for the latency-reactivation cycle. Several studies have also concluded that LAT inhibits productive infection. To date, a functional LAT protein has not been identified, suggesting that LAT is a regulatory RNA. Two small RNAs (sRNAs) were previously identified within the first 1.5 kb of LAT coding sequences. In this study, we demonstrated that both LAT sRNAs were expressed in the trigeminal ganglia of mice latently infected with an HSV-1 strain that expresses LAT but not when mice were infected with a LAT null mutant. LAT sRNA1 and sRNA2 cooperated to inhibit cold shock-induced apoptosis in mouse neuroblastoma cells. LAT sRNA1, but not LAT sRNA2, inhibited apoptosis less efficiently than both sRNAs. When rabbit skin cells were cotransfected with plasmids that express LAT sRNA1 and HSV-1 genomic DNA, the amount of infectious virus released was reduced approximately 3 logs. Although LAT sRNA2 was less effective at inhibiting virus production, it inhibited expression of infected cell protein 4 (ICP4). Neither LAT sRNA had an obvious effect on ICP0 expression. These studies suggested that expression of two LAT sRNAs plays a role in the latency-reactivation cycle by inhibiting apoptosis and productive infection.

Neurological disease in cattle in southern Brazil associated with Bovine herpesvirus infection.

The occurrence of neurological disease in cattle caused by Bovine herpesvirus in 11 farms from southern Brazil between 1987 and 2007 is described. Twenty-two animals were necropsied. Major clinical signs included excessive salivation, nasal and ocular discharge, circling, recumbency, depression, incoordination, grinding of teeth, and paddling movements. Necropsy findings in 10 of 22 cattle included hyperemia and softening of the rostral portions of the telencephalic cortex, with flattening of gyri, and malacia. Cattle in 10 cases did not show any gross lesions. Histological examination in most cases revealed nonsuppurative and necrotizing meningoencephalitis with acute neuronal necrosis, edema, eosinophilic intranuclear inclusion bodies in astrocytes and neurons, and infiltration of gitter cells. No histologic lesions could be detected in 4 cases. The initial diagnosis was based upon the clinical, epidemiological, and pathological findings. The diagnosis was confirmed by virus isolation in cell culture followed by virus identification by a glycoprotein C-based polymerase chain reaction. Seven isolates were identified as Bovine herpesvirus 5, and 4 were identified as Bovine herpesvirus 1.

Phenotypical assays and partial sequencing of the hsp60 gene for identification of Streptococcus equi.

Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of strangles by phenotypic tests and to analyze the partial sequences obtained from fragments of the hsp60 gene. In this work, 26 strains of Streptococcus spp. isolated from horse clinical samples were analyzed. By phenotypical assays, 18 were characterized as S. equi subsp. equi, five as S. equi subsp. zooepidemicus, two as S. dysgalactiae subsp. equisimilis, and one as Streptococcus sp. However 21 isolates were identified as S. equi subsp. equi and five as S. equi subsp. zooepidemicus by DNA sequencing. The sequencing of the partial hsp60 gene was demonstrated to be an alternative method to analyze and differentiate strains of Streptococcus spp. In addition, this method can be useful as a discriminatory tool for characterization of atypical isolates.