Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor.

UNLABELLED: Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from <3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14(+) monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus. IMPORTANCE: Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein).

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

A retrospective study on the epidemiology of anthrax, foot and mouth disease, haemorrhagic septicaemia, peste des petits ruminants and rabies in Bangladesh, 2010-2012.

Anthrax, foot and mouth disease (FMD), haemorrhagic septicaemia (HS), peste des petits ruminants (PPR) and rabies are considered to be endemic in Bangladesh. This retrospective study was conducted to understand the geographic and seasonal distribution of these major infectious diseases in livestock based on data collected through passive surveillance from 1 January 2010 to 31 December 2012. Data analysis for this period revealed 5,937 cases of anthrax, 300,333 of FMD, 13,436 of HS, 247,783 of PPR and 14,085 cases of dog bite/rabies. While diseases were reported in almost every district of the country, the highest frequency of occurrence corresponded to the susceptible livestock population in the respective districts. There was no significant difference in the disease occurrences between districts bordering India/Myanmar and non-border districts (p>0.05). Significantly higher (p<0.01) numbers of anthrax (84.5%), FMD (88.3%), HS (84.9%) and dog bite/rabies (64.3%) cases were reported in cattle than any other species. PPR cases were reported mostly (94.8%) in goats with only isolated cases (5.2%) in sheep. The diseases occur throughout the year with peak numbers reported during June through September and lowest during December through April, with significant differences (p<0.01) between the months. The annual usages of vaccines for anthrax, FMD, HS and PPR were only 7.31%, 0.61%, 0.84% and 11.59% of the susceptible livestock population, respectively. Prophylactic vaccination against rabies was 21.16% of cases. There were significant differences (p<0.01) in the administration of anthrax, FMD and HS vaccines between border and non-border districts, but not PPR or rabies vaccines. We recommend that surveillance and reporting of these diseases need to be improved throughout the country. Furthermore, all suspected clinical cases should be confirmed by laboratory examination. The findings of this study can be used in the formulation of more effective disease management and control strategies, including appropriate vaccination policies in Bangladesh.

Sequence analysis of infectious bronchitis virus isolates from the 1960s in the United States.

To better understand the molecular epidemiology of infectious bronchitis virus (IBV) in the United States following the introduction of commercial IBV vaccines, we sequenced the S1 and N structural protein genes of thirteen IBV field isolates collected in the 1960s. Analysis of the S1 sequence showed that seven isolates were of the Massachusetts (Mass) genotype, five were SE17, and one was of the Connecticut (Conn) genotype, suggesting that these three IBVs were circulating in commercial poultry raised in different regions in the United States during the 1960s. The S1 genes of Mass-type isolates had high levels of sequence variation, representing 81.3-81.9 % nucleotide (nt) and 77.3-78.7 % amino acid (aa) identity when compared to those of the SE17-type isolates. In contrast, the N genes from the same isolates were less variable (>92 % nt and >93 % aa identity) when compared to those of the SE17-type isolates. Phylogenetic analysis based on the S1 gene indicated that one isolate (L748) was more closely related to the Mass type. In contrast, phylogenetic analysis based on the N gene showed that L748 was more closely related to the SE17 type, indicating that there had been exchange of S1 genetic materials between Mass- and SE17-like viruses. In addition, the Mass-type isolates had high levels of sequence identity in the S1 gene compared with widely used modified live vaccines (Mass41, Ma5 and H120) and modern field strains from the USA and other countries, suggesting a common ancestor.

A comparison of virulence of influenza A virus isolates from mallards in experimentally inoculated turkeys.

Low pathogenic avian influenza viruses (LPAIV) from wild waterfowl can and do cross species barriers, infecting and sometimes becoming established in domestic poultry. Turkeys are naturally highly susceptible to LPAIV infections, especially with viruses from ducks. In this study, we describe clinical signs and lesions in experimentally inoculated commercial turkeys produced by a LPAIV, A/mallard/MN/1714/09 (H7N1), isolated from a mallard duck. Our results demonstrate that this H7N1 isolate produced clinical signs, including severe edema of the head and face because of an early inflammatory response in both inoculated and contact turkeys. In comparison, an isolate, A/mallard/MN/2749/09 (H6N8) from the same mallard population, infected and was transmitted between naive turkeys but did not cause clinical disease or lesions. Our data indicate that proinflammatory (IL-1beta, TNF-alpha, and IL-6) and antiviral (IFN-gamma and IL-2) cytokines are expressed at different levels in H7N1- and H6N8-infected turkey peripheral blood mononuclear cells. These differences correlate inversely with clinical lesions, suggesting that differences in host responses result in variances in viral pathogenesis and in virulence of LPAIV in commercial turkeys. Based on these results, we can conclude that turkeys may exhibit variable immunologic responses to infection with different AIV strains.

Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum sanctum Linn.) leaf extract on healthy volunteers.

ETHNOPHARMACOLOGICAL RELEVANCE: Tulsi (Ocimum sanctum Linn.) is considered as a sacred herb and traditionally it is believed that consumption of Tulsi leaf on empty stomach increases immunity. Experimental studies have shown that alcoholic extract of Tulsi modulates immunity. MATERIALS AND METHODS: The present study was designed to evaluate the immunomodulatory effects of ethanolic extract of Tulsi leaves through a double-blinded randomized controlled cross-over trial on healthy volunteers. Three hundred milligrams capsules of ethanolic extracts of leaves of Tulsi or placebo were administered to 24 healthy volunteers on empty stomach and the results of 22 subjects who completed the study were analyzed. The primary objective was to study the levels of Th1 and Th2 cytokines (interferon-γ and interleukin-4) during both pre and post intervention period in blood culture supernatants following stimulation with lipopolysaccharide and phytohaemagglutinin. Other immunological parameters such as T-helper and T-cytotoxic cells, B-cells and NK-cells also were analyzed using Flowcytometry. RESULTS: Statistically significant increase in the levels of IFN-γ (p=0.039), IL-4 (p=0.001) and percentages of T-helper cells (p=0.001) and NK-cells (p=0.017) were observed after 4 weeks in the Tulsi extract intervention group in contrast to the placebo group. CONCLUSIONS: These observations clearly ascertain the immunomodulatory role of Tulsi leaves extract on healthy volunteers.

Role of Marek's disease herpesvirus in the induction of tumours in Japanese quail (Coturnix coturnix japonica) by methylcholanthrene.

The QT35 cell line, established from 20-methylcholanthrene (MCA)-induced tumours in Japanese quail, is positive for Marek's disease virus (MDV), and therefore we examined whether MDV is important for the development of MCA-induced tumours. Japanese quail were inoculated with the JM16 strain of MDV at 1 or 3 days of age or left uninoculated. At 3 weeks of age, quail were injected in the breast muscle with 4 mg MCA in corn oil or corn oil alone. Quail were observed for tumours three times/week and at post mortem at 11 to 12 weeks of age. MDV DNA was detected by polymerase chain reaction (PCR) in spleens of 14/20 birds inoculated with JM16+corn oil and of 53/71 birds inoculated with JM16+MCA. Interestingly, 1/74 quail was positive in the MCA group alone for MDV DNA. Tumours were collected for histopathology, cell line development, and PCR and reverse transcriptase-PCR for the presence of MDV. Tumours developed in 38/83 MCA-treated and 32/85 JM16+MCA-treated quail. Fibrosarcomas without metastasis were the only tumours observed in the MCA-treated quail, while quail treated with JM16 and MCA developed undifferentiated tumours, fibrosarcomas, lymphosarcomas or combinations with or without metastasis. One out of 20 quail receiving JM16 alone developed a lymphosarcoma. Cell line development was not influenced by JM16. Tumours from MCA-treated quail were negative for MDV, while 19/29 were positive in the JM16+MCA group. MDV transcripts were present in 13/18 tumours examined in the JM16+MCA group. In conclusion, MDV did not affect tumour development but did influence tumour aggression and histological type.

The science behind sacredness of Tulsi (Ocimum sanctum Linn.).

Medicinal properties of Tulsi (Ocimum sanctum Linn) are known for thousand years to various civilizations of the world. This medicinal herb is considered as a sacred plant by the Hindus in the Indian subcontinent. Scientific explorations of traditional belief of medicinal properties of Tulsi have got momentum mostly after the middle of the 20th century. In the present review, efforts have been made to sum up different aspects of scientific studies on this medicinal plant. Scientific evidences are available on various medicinal aspects i.e. antimicrobial, adaptogenic, antidiabetic, hepato-protective, anti-inflammatory, anti-carcinogenic, radioprotective, immunomodulatory, neuro-protective, cardio-protective, mosquito repellent etc. to name a few. Most of these evidences are based on in-vitro, experimental and a few human studies.

Molecular characterization of a poxvirus isolated from an American flamingo (Phoeniconais ruber rubber).

An avian poxvirus from the beak scab of an American flamingo (Phoeniconais ruber rubber) was isolated by inoculation on the chorioallantoic membrane (CAM) of specific-pathogen-free (SPF) chicken embryos. The virus produced multifocal areas of epithelial hyperplasia along with foci of inflammation in the CAM, and rare cells contained small eosinophilic intracytoplasmic bodies. Chickens inoculated with the isolated virus in the feather follicle of the leg did not develop significant lesions. Nucleotide sequence comparison of a PCR-amplified 4.5 kb HindIII fragment of the genome of flamingo poxvirus (FlPV) revealed very high homology (99.7%) with condor poxvirus (CPV), followed by approximately 92% similarity with canary poxvirus (CNPV) and Hawaiian goose poxvirus (HGPV), but less similarity (approximately 69%) to fowl poxvirus (FPV), the type species of the genus Avipoxvirus of family Poxviridae. As in the cases with CPV, CNPV, and HGPV, genetic analysis of FlPV revealed an absence of three corresponding FPV open reading frames (ORF199, 200, and 202) and an absence of any reticuloendotheliosis virus (REV) sequences in this region. There are only nine nucleotide substitutions observed between FlPV and CPV in the 4.5 kb fragment; those were clustered in the ORF201 region, which in FPV genome is a site for integration of REV sequences. Phylogenetic analysis of the predicted amino acid sequences of the ORF201-coded hypothetical protein demonstrated FlPV to be more closely related to CPV, as well as to CNPV and HGPV, than to FPV.

Genotypic and phenotypic characterization of the California 99 (Cal99) variant of infectious bronchitis virus.

The California 99 (Cal99) variant of infectious bronchitis virus (IBV) was first recovered in 1999 from vaccinated broiler chicken flocks in Central California. The S1 hypervariable region of Cal99 genome was most closely related to Arkansas (Ark) serotype viruses. In this study, the complete genome of Cal99 was sequenced, and the structural protein genes were compared with those of commonly used IBV vaccines as well as those of isolates from naturally occurring outbreaks in different parts of the world, to elucidate potential sources of genetic material. Based on sequence comparison, the prototype Cal99 virus is similar to the apathogenic ArkDPI virus, except in the S1 gene and stretches of sequence in the S2 and M structural protein genes, which are more related to Connecticut (Conn) and Massachusetts (Mass) strain viruses, respectively. We speculate that these two fragments came from a Conn and a Mass virus, respectively, and were incorporated into a virus largely derived from ArkDPI. Since Ark, Conn and Mass strains have been simultaneously used as live vaccines in California, both point mutations and recombination among vaccine strains may have contributed to the emergence of the Cal99 variant virus. Analysis of the structural protein genes of six Cal99 isolates demonstrated that viruses of this serotype may differ substantially in the non-S1 structural genes. Finally, we performed a challenge study with Cal99 and demonstrated that the virus causes late-onset respiratory disease, with a severity comparable to that of the M41 IBV challenge strain.

Comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains.

Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5' end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PL(pro)), main protease (M(pro)), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase gene are important for virus replication. We have sequenced four regions of the replicase genes corresponding to the 5'-terminal sequence, PL(pro), M(pro), and RdRp domains from 20 heterologous IBV strains, and compared them with previously published coronavirus sequences. All the coronavirus 5'-termini and PL(pro) domains were divergent, unlike the M(pro) and the RdRp domains that were highly conserved with 28% and 48% conserved residues, respectively. Among IBV strains, the 5' untranslated region including the leader sequence was highly conserved (>94% identical); whereas, the N-terminal coding region and the PL(pro) domains were highly variable ranging from 84.6% to 100%, and 77.6% to 100% identity, respectively. The IBV M(pro) and RdRp domains were highly conserved with 82.7% and 92.7% conserved residues, respectively. The BJ strain was the most different from other IBVs in all four regions of the replicase. Phylogeny-based clustering based on replicase genes was identical to the antigen-based classification of coronaviruses into three groups. However, the IBV strain classification based on replicase gene domains did not correlate with that of the type-specific antigenic groups. The replicase gene sequences of many IBVs recovered from infected chickens were identical to those of vaccine viruses irrespective of serotype, suggesting that either there has been an exchange of genetic material among vaccine and field isolates or that there is a convergent evolution to a specific replicase genotype. There was no correlation between the genotype of any region of the replicase gene and pathotype, suggesting that the replicase is not the sole determinant of IBV pathogenicity.

Establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens.

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant disease in chickens. Establishment of a carrier state in IBV infection and the potential for the persistent virus to undergo mutations and recombination in chicken tissues have important consequences for disease management. Nevertheless, whether chickens can maintain persistent IBV infection in the absence of reinfection from exogenous sources or the presence of antibody in the host can modulate virus persistence remains unclear. Indeed, whether or not IBV genome can undergo genetic changes during in vivo infection has not been demonstrated experimentally. In the present study, IBV shedding and tissue persistence were monitored in individual chickens maintained under strict isolation that precluded reinfection from exogenous sources. In the first of two experiments, intranasal exposure of 6-wk-old antibody-free chickens to IBV vaccine virus resulted in intermittent shedding of the virus from both trachea and cloaca of individual birds for up to 63 days. Also, the virus was recovered from the internal organs (spleen, gonad, kidney, lung, cecal tonsil, and cloacal bursa) of six of eight birds killed at various intervals between 27 and 163 days postinoculation (DPI). In the second experiment, IBV exposure of 1-day-old maternal antibody-positive chicks led to periodic virus shedding from the trachea and cloaca in all chickens until 77 days; however, internal organs (lungs and kidneys) of only one of seven birds (killed at 175 DPI) were virus positive, suggesting that presence of antibody at the time of infection protects internal organs from IBV infection. When the lung and kidney isolates of IBV from the latter experiment were compared with the parent-vaccine virus, no changes in their antigenicity, tissue tropism, or the nucleotide sequence of the S1 glycoprotein gene were observed. These findings indicate that, unlike the mammalian coronaviruses, propensity for frequent genetic change may not be inherent in the IBV genome.

Characterization of infectious bronchitis virus isolates by slot blot hybridization.

We used slot blot hybridization of the hypervariable regions of the S1 subunit of spike peplomer gene to identify and characterize infectious bronchitis virus (IBV) strains. Template DNA was created from six reference strain IBVs of different serotypes and immobilized on a nitrocellulose membrane. We synthesized digoxigenin-labeled probes from reference and unknown field viruses and hybridized them to template DNA. All reference strains could be distinguished and isolates identified by serotype if they were at least 95% identical to a reference strain. This slot blot hybridization procedure was specific and reproducible, and strain typing was consistent with the S1 sequencing of the IBV genome. This study thus provides a simple and rapid method for typing of IBV.