A novel point mutation (L70P) inactivates poliovirus 3C protease.

Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two ß-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

Expression and purification of polioviral proteins in E. coli, and production of antisera as reagents for immunological assays.

Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in Escherichia coli using the autoinduction system, purified, and the proteins were used to raise antisera in rabbits. All antisera detected all three serotypes of PV from infected cell lysates in enzyme-linked immunosorbent assay, immunofluorescence and western blotting.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.

Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.

Dual Infection with Bluetongue Virus Serotypes and First-Time Isolation of Serotype 5 in India.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks.

Epidemiology of Bluetongue in India.

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.

Molecular Typing of Bluetongue Viruses Isolated Over a Decade in South India.

Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines.

Isolation of Bluetongue Virus 24 from India - An Exotic Serotype to Australasia.

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

Inhibition of the MHC class II antigen presentation pathway by human cytomegalovirus.

Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals. Normally, anti-HCMV immune response controls virus replication following reactivation from latency. However, HCMV, like other large herpesviruses, encodes immune evasion proteins that allow the virus to replicate, for a time or in specific tissues, and produce viral progeny in the face of robust host immunity. HCMV glycoproteins US2, US3, US6 and US11 all inhibit different stages of the MHC class I antigen presentation pathway and can reduce recognition by CD8+ T lymphocytes. Here, we discuss two novel inhibitors of the MHC class II antigen presentation pathway, HCMV glycoproteins US2 and US3. Both US2 and US3 can inhibit presentation of exogenous protein antigens to CD4+ T lymphocytes in in vitro assays. US2 causes degradation of MHC class II molecules: HLA-DR-alpha and HLA-DM-alpha, as well as class I heavy chain (HC), but does not affect DR-beta or DM-beta chains. Mutant forms of US2 have been constructed that can bind to DR-alpha and class I HC but do not cause their degradation, separating the binding step from other processes that precede degradation. We also found evidence that US2-induced degradation of class I and II proteins involves a cellular component, other than Sec61, that is limiting in quantity. Unlike US2, US3 binds newly synthesized class II alpha/beta complexes, reducing the association with the invariant chain (Ii) and causing mislocalization of class II complexes in cells. US3 expression reduces accumulation of class II complexes in peptide-loading compartments and loading of peptides. Since US2 and US3 are expressed solely within HCMV-infected cells, it appears that these viral proteins have evolved to inhibit presentation of endogenous, intracellular viral antigens to anti-HCMV CD4+ T cells. This is different from how the MHC class II pathway is normally viewed, as a pathway for presentation of exogenous, extracellular proteins. The existence of these proteins indicates the importance of class II-mediated presentation of endogenous antigens in signalling virus infection to CD4+ T cells.

Isolation and Complete Genome Sequencing of Bluetongue Virus Serotype 12 from India.

Bluetongue virus (BTV) causes disease mainly in sheep, but can be transmitted via other domestic and wild ruminants, resulting in pecuniary burden and trade restrictions. Segmented genome with the possibility of reassortment, existence of 26 serotypes, geographical restriction in the distribution of many of the serotypes, use of live attenuated vaccines and the lack of complete sequences of viruses isolated from several parts of the globe have complicated our understanding of the origin, movement and distribution of BTV. Recent efforts in genome sequencing of several strains have helped in better comprehending BTV epidemiology. In an effort to contribute to the genetic epidemiology of BTV in India, we report the isolation and complete genome sequencing of a BTV serotype 12 virus (designated NMO1). This is the first BTV-12 isolated from India and the second BTV-12 to be sequenced worldwide. The analysis of sequences of this virus suggests that NMO1 derived its segments from viruses belonging to western topotype viruses, as well as those from South-East Asia and India. The results have implications for understanding the origin, emergence/re-emergence and movement of BTV as well as for the development of vaccines and diagnostics based on robust epidemiological data.

Typing of Staphylococcus aureus isolated from bovine mastitis cases in Australia and India.

OBJECTIVE: To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India. METHODS: A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR. RESULTS: Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively. CONCLUSIONS: Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis.

Bovine lymphocyte antigen-A11--specific peptide motif as a means to identify cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present 8- to 10-mer viral peptides to antiviral cytotoxic T lymphocytes (CTLs). Identification of the allele-specific peptide motifs (ASPMs) of class I molecules enables the prediction of potential CTL epitopes of a virus from its protein sequences. Based on the bovine herpesvirus 1 (BHV-1) protein sequences that conform to the BoLA-A11 ASPM that we identified previously, potential CTL epitopes of BHV-1 were synthesized for use in cytotoxicity assays with CTLs from BHV-1-immunized calves. A peptide binding assay used to select the peptides that are most likely to be CTL epitopes categorized the peptides into groups of high, intermediate, and low binding capacity. Synthetic peptides stimulated lymphocytes from BHV-1-immunized calves to secrete interferon-gamma. Groups of peptides from the major glycoproteins of BHV-1 restimulated CTLs in vitro and sensitized targets for lysis by means of restimulated bulk CTLs.

Development of a syngeneic bovine fibroblast cell line: implications for the study of bovine cytotoxic T lymphocytes.

The study of T-cell-mediated cytotoxicity in domestic animals, especially in cattle, has been hampered by the lack of proper restimulatory as well as target systems. While the currently available bovine cell lines have not been typed for the major histocompatibility complex (MHC) class I molecules they express, methods to derive lines of cells obtained from animals that are MHC-typed have not been thoroughly explored. In the present study, we describe a method for the development of cell lines from MHC-typed animals. Cells obtained from the skin of a calf typed as bovine lymphocyte antigen-A11/-A13 were transfected with a plasmid containing the whole genome of simian vacuolating virus 40 (SV40). A cell line was derived from the resultant transfectants. This cell line expressed bovine MHC class I molecules on the cell surface, and SV40 large T antigen in the nucleus. The cells were permissive to the replicative cycle of bovine herpesvirus-1 (BHV-1), and the major glycoproteins of BHV-1 were expressed at expected times after infection. The present study should contribute to the study of cytotoxic T lymphocyte response of cattle to BHV-1 and other intracellular pathogens.

Reverse immunogenetic and polyepitopic approaches for the induction of cell-mediated immunity against bovine viral pathogens.

The control of several infectious diseases of animals by vaccination is perhaps the most outstanding accomplishment of veterinary medicine in the last century. Even the eradication of some pathogens is in sight, at least in some parts of the world. However, infectious diseases continue to cost millions of dollars to the livestock industry. One of the reasons for the failure to control certain pathogens is the limited emphasis placed on cell-mediated immunity (CMI) in the design of vaccines against these pathogens. Traditionally, vaccine-induced immunity has been studied in relation to antibody-mediated protection. More recent studies, however, have focused on understanding CMI and developing means of inducing CMI. This review focuses on recent advances made in the study of CMI in general and of cytotoxic T lymphocytes in particular. Parallels from studies in human and mouse immunology are drawn in order to point out implications to bovine immunology, specifically for immunity against bovine herpesvirus 1.

Sequences of genes encoding type-specific and group-specific antigens of an Indian isolate of bluetongue virus serotype 10 (BTV-10) and implications for their origin.

Bluetongue, a transboundary disease, is endemic in several tropical countries and is caused by bluetongue virus (BTV). The origin and movement of BTV can be predicted by comparing nucleotide sequences of its segmented RNA genome. Such analyses have been useful in evaluating the source of the virus responsible for recent incursion of BTV into previously unreported areas. Besides several serotypes, genetically related BTV strains circulate in each endemic area, but such clusters of strains have been reported to be distinct from one geographical region to another. We obtained partial or complete sequences of the open reading frames encoded by VP2, VP6, VP7, NS1 and NS2 genes of a BTV-10 isolate of India and compared them with other BTV-10 sequences available in public database. Sequences of all the five genes showed >99% identity to BTV-10 prototype, vaccine strain and vaccine-like virus isolates from the USA. Our results suggest that Indian BTV-10 virus analysed in this study possibly originated from the United States.

The use of bovine MHC class I allele-specific peptide motifs and proteolytic cleavage specificities for the prediction of potential cytotoxic T lymphocyte epitopes of bovine viral diarrhea virus.

Cell mediated immunity (CMI) is crucial for the defense against viruses. Cytotoxic T lymphocytes (CTLs) play a major role in CMI. They recognize endogenous antigenic peptides presented by antigen presenting cells in association with the major histocompatibility complex (MHC) class I molecules. The elucidation of the sequence of CTL epitopes of viruses should help in designing better vaccines. In this study, we have identified candidate epitopes restricted by five bovine MHC class I molecules that are potentially available for presentation to CTLs. The candidate peptide epitopes were identified by using the computer programs available as a part of the Genetics Computer Group package and applying the information on allele-specific peptide motifs and intracellular enzymatic cleavage patterns to the bovine viral diarrhea virus polyprotein. Several candidate peptides were found for each of the bovine lymphocyte antigens (BoLA)-A11, -A20, -HD1, and -HD6 whereas no peptide was found for BoLA-HD7. Based on this finding, the probable contribution of genomic segments of BVDV to the CTL response and strategies for recombinant vaccines are discussed.

Prediction of potential cytotoxic T lymphocyte epitopes of bovine herpesvirus 1 based on allele-specific peptide motifs and proteolytic cleavage specificities.

Major histocompatibility complex (MHC) class I molecules present endogenous peptides to cytotoxic T lymphocytes (CTLs). Elucidation of CTL epitopes of intracellular pathogens helps in designing better vaccines to control economically important human and animal diseases. In this study, candidate epitopes that are potentially available for presentation to the CTLs via five bovine MHC class I molecules have been identified. This was accomplished by using the computer programs "Find-patterns" and "Peptidestructure" of GCG package and applying the information on cleavage patterns of cytosolic and endoplasmic reticulum proteases and peptidases as well as MHC class I allele-specific peptide motifs on 23 bovine herpesvirus-1 (BHV-1) proteins available on protein sequence database. Several candidate peptides were found for each of the bovine lymphocyte antigens (BoLA)-A11, -A20, -HD1, and -HD6 whereas no peptide was found for BoLA-HD7. Majority of the candidate peptides were from the viral glycoproteins. The contribution of such studies towards the identification of CTL epitopes of BHV-1 and other intracellular pathogens is discussed.

Identification of murine cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present endogenously derived viral peptides to CD8+ cytotoxic T-lymphocytes (CTLs). The objective of this study was to identify the H-2Dd- and H-2Kd-restricted CTL epitopes of bovine herpesvirus 1 (BHV-1), based on the allele-specific peptide motifs (ASPMs) of the above class I molecules. Nine sequences conforming to the H-2Dd and H-2Kd ASPMs were identified on BHV-1 proteins, and the respective peptides were synthesized. Five of these peptides exhibited moderate to strong binding to the Dd molecule. CTLs generated by BALB/c mice immunized with BHV-1 proteins emulsified in a suitable adjuvant effectively lysed peptide-pulsed syngeneic targets, indicating that these epitopes were generated in vivo. Mice immunized with these peptides emulsified in a suitable adjuvant also developed anti-BHV-1 CTLs. These CTLs identified three veritable CTL epitopes among the "potential epitopes" synthesized based on the ASPMs. The elucidation of the CTL epitopes of BHV-1 should aid in the development of efficacious vaccines against this virus.

Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes.

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.