Hesperetin blocks poxvirus replication with a low tendency to select for drug-resistant viral variants.

In this study, we demonstrated the antiviral efficacy of hesperetin against multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus (VACV), and lumpy skin disease virus (LSDV). The time-of-addition and virus step-specific assays indicated that hesperetin reduces the levels of viral DNA, mRNA, and proteins in the target cells. Further, by immunoprecipitation (IP) of the viral RNA from BPXV-infected Vero cells and a cell-free RNA-IP assay, we demonstrated that hesperetin-induced reduction in BPXV protein synthesis is also consistent with diminished interaction between eukaryotic translation initiation factor eIF4E and the 5' cap of viral mRNA. Molecular docking and MD simulation studies were also consistent with the binding of hesperetin to the cap-binding pocket of eIF4E, adopting a conformation similar to m7GTP binding. Furthermore, in a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane and associated mortality in the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we, for the first time, demonstrated the antiviral activity of hesperetin against multiple poxviruses, besides providing some insights into its potential mechanisms of action.

Buffalopox Disease in Livestock and Milkers, India.

Buffalopox outbreaks caused by vaccinia virus were observed in villages of Tamil Nadu, India, among lactating buffaloes and cows. Milkers also had lesions on their fingers. Because vaccinia virus is known to have extended its host range in Brazil, we recommend continuous surveillance to understand cross-species transmission and to curtail disease effects.

The Disease Ecology, Epidemiology, Clinical Manifestations, Management, Prevention, and Control of Increasing Human Infections with Animal Orthopoxviruses.

Zoonotic orthopoxvirus outbreaks have occurred repeatedly worldwide, including monkeypox in Africa and the United States, cowpox in Europe, camelpox in the Middle East and India, buffalopox in India, vaccinia in South America, and novel emerging orthopoxvirus infections in the United States, Europe, Asia, and South America. Waning smallpox immunity may increase the potential for animal-to-human transmission followed by further community transmission person-to-person (as demonstrated by monkeypox and buffalopox outbreaks) and by contact with fomites (as demonstrated by camelpox, cowpox, and, possibly, Alaskapox). The objectives of this review are to describe the disease ecology, epidemiology, clinical manifestations, prevention, and control of human infections with animal orthopoxviruses and to discuss the association with diminished population herd immunity formerly induced by vaccinia vaccination against smallpox. Internet search engines were queried with key words, and case reports, case series, seroprevalence studies, and epidemiologic investigations were found for review.

Comparative efficacy of chemical stabilizers on the thermostabilization of a novel live attenuated buffalopox vaccine.

In the present investigation, the thermostability of a live attenuated buffalopox vaccine prepared with an indigenous baffalopox virus isolate (BPXV Vij/96) and freeze-dried under conventional lyophilizing conditions is described. Three different stabilizer combinations like LS (lactalbumin hydralysate + sucrose), LHT (lactalbumin hydralysate + Trehalose dihydrate) and TAA (Trehalose dihydrate + l- Alanine + l-Histidine) were used to prepare the vaccine. The study indicated that the LS stabilizer was found to be the stabilizer of choice followed by LHT and TAA for buffalopox vaccine at all temperatures studied. The presence of stabilizers has beneficial influence in preserving the keeping quality of the vaccine. Further, among the diluents used to reconstitute the freeze-dried buffalopox vaccine, double distilled water, 0.85% normal saline solution and phosphate buffer saline were the choice of diluents in that order. However, 1M MgSO4 did not perform well at higher temperatures. Investigation suggests for using LS as a stabilizer for freeze-drying and any of the three diluents except 1MgSO4 for reconstitution of buffalopox vaccine.

Detection of Vaccinia Virus in Dairy Cattle Serum Samples from 2009, Uruguay.

We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out.

Buffalopox: An emerging zoonotic challenge.

As a variant of Vaccinia virus, Buffalopox virus is known to cause Buffalopox disease. In recent times, sporadic outbreaks of the infection in humans have been reported, especially in the endemic countries of Southeast Asia. Though mortality has not been high, associated morbidity is significant. Due to waning cross-protective immunity against smallpox, Buffalopox virus is one of the several orthopox viruses likely to emerge or reemerge. To combat this virus, early recognition, isolation, and management of the infection in animals and humans is of prime importance. In addition, vaccination in animals and humans at risk of acquiring infection is essential as a means of limiting animal-to-animal and animal-to-human spread of the virus. With this in mind, a collaborative approach between the animal and human health sectors is indispensable.

Comparative Pathology of Zoonotic Orthopoxviruses.

This review provides a brief history of the impacts that a human-specific Orthopoxvirus (OPXV), Variola virus, had on mankind, recalls how critical vaccination was for the eradication of this disease, and discusses the consequences of discontinuing vaccination against OPXV. One of these consequences is the emergence of zoonotic OPXV diseases, including Monkeypox virus (MPXV). The focus of this manuscript is to compare pathology associated with zoonotic OPXV infection in veterinary species and in humans. Efficient recognition of poxvirus lesions and other, more subtle signs of disease in multiple species is critical to prevent further spread of poxvirus infections. Additionally included are a synopsis of the pathology observed in animal models of MPXV infection, the recent spread of MPXV among humans, and a discussion of the potential for this virus to persist in Europe and the Americas.

Buffalopox Virus: An Emerging Virus in Livestock and Humans.

Buffalopox virus (BPXV) is the cause of buffalopox, which was recognized by the FAO/WHO Joint Expert Committee on Zoonosis as an important zoonotic disease. Buffalopox was first described in India, later in other countries, and has become an emerging contagious viral zoonotic disease infecting milkers with high morbidity among affected domestic buffalo and cattle. BPXV is a member of the genus Orthopoxvirus and a close variant of the vaccinia virus (VACV). Recent genome data show that BPXV shares a most recent common ancestor of VACV Lister strain, which had been used for inoculating buffalo calves to produce a Smallpox vaccine. Over time, VACV evolved into BPXV by establishing itself in buffaloes to be increasingly pathogenic to this host and to make infections in cattle and humans. Together with the current pandemic of SARS-COV2/COVID 19, BPXV infections illustrate how vulnerable the human population is to the emergence and re-emergence of viral pathogens from unsuspected sources. In view that majority of the world population are not vaccinated against smallpox and are most vulnerable in the event of its re-emergence, reviewing and understanding the biology of vaccinia-like viruses are necessary for developing a new generation of safer smallpox vaccines in the smallpox-free world.

Antiviral activity of Apigenin against buffalopox: Novel mechanistic insights and drug-resistance considerations.

We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.

Isolation and phylogenomic analysis of buffalopox virus from human and buffaloes in India.

Three genome sequences of Buffalopox virus (BPVX) were retrieved from a human and two buffaloes scab samples. Phylogenomic analysis of the BPXV indicates that it shares a most recent common ancestor with Lister and closely related vaccine strains when compared to potential wild-type VACV strains (like Horsepox virus).

An Update on the Known Host Range of the Brazilian Vaccinia Virus: An Outbreak in Buffalo Calves.

Even nearly forty years after the eradication of smallpox, members of the Poxviridae family continue to be the focus of an increasing number of studies. Among these studies, prominently stands vaccinia virus, an orthopoxvirus that is associated with bovine vaccinia outbreaks. Although more frequently associated with infections in cattle and humans, the host range of vaccinia virus is not restricted only to these hosts. There are several instances of molecular and serological evidence of circulation of vaccinia virus among wildlife species. In addition, viral isolation has confirmed a broad spectrum of vaccinia virus hosts. In this report, we provide a brief update on the host range of Brazilian vaccinia virus, and present a case description of an outbreak in domestic buffalo calves from Northeastern Brazil that corroborates previous serological and molecular studies. Furthermore, in the present study, vaccinia virus has been isolated for the first time in buffaloes, and referred to as vaccinia virus Pernambuco (VACV-PE). Phylogenetic reconstruction was based on A56R clustered VACV-PE with vaccinia virus isolates belonging to group 1 Brazilian vaccinia virus. Furthermore, the vaccinia virus genome was detected in the milk of a lactating cow, which thereby revealed a pathway for future studies on the possible impact of vaccinia virus on buffalo milk and milk products. Taken together, these results provide the first description of clinical disease caused by vaccinia virus in buffaloes in South America. They also raise new questions about the chain of transmission of this virus.

Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L (21S to E110; ∼30 kDa) and rH3L(1M to I280; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 µg of each) was used for active immunization of adult mice (20µg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV.

Immunogenicity and protective efficacy of recombinant major envelope protein (rH3L) of buffalopox virus in animal models.

Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 µg/dose) and adult mice (10 µg and 50 µg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox.

Structural analysis and immunogenicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia-like virus.

Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 µg/rabbit) and adult mice (10 µg and 50 µg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox.

Emergence and reemergence of vaccinia-like viruses: global scenario and perspectives.

Among the members of the genus Orthopoxvirus (OPXV), vaccinia virus (VACV), the type species of the genus is a double-stranded DNA virus, belongs to the subfamily Chordopoxvirinae of the family Poxviridae. The causative agents of smallpox, VACV and Variola virus are mutually immunogenic and the type species of Orthopoxvirus, cause only mild complications in humans. Therefore, the VACV was used as a smallpox vaccine world over under mass immunization program promoted by World Health Organization, which lead to the variola eradication globally in 1979. Since then, no vaccination of human population has been carried out; however, vaccination has been continued for at-risk laboratory workers, military personnel and others working with recombinant VACV or other non-variola orthopoxviruses (OPXVs). There has now been a surge in the development of safer smallpox vaccines and understanding of the biology of VACV necessitating re-use of this vaccine in most vulnerable population, because of rise in bioterrorist threats globally. Also, globally there has been the emergence and re-emergence of vaccinia-like viruses (VLVs) in Brazil, buffalopox viruses in Egypt, Indonesia, India and its neighbouring countries like Nepal, Pakistan. Bioterrorism as well as emergence and re-emergence of the VLVs constitute a concern as 50 % of the population globally (40 % in USA) <30 years are unvaccinated and most vulnerable for smallpox reemergence. Thus, the search for new generation safer smallpox vaccine entails review of biology of VLVs in the smallpox-free world. In this review, we present occurrence of VLVs in the world with exhaustive discussion particularly on the emergence and re-emergence of these viruses in India and Brazil where VLVs are sufficiently studied.

Analgesic, Antibacterial and Antiviral Activities of 2-(5-Alkyl-1,3,4-oxadiazol-2-yl)-3H-benzo[f]chromen-3-ones.

A novel series of 2-(5-alkyl-1,3,4-oxadiazol-2-yl)-3H-benzo[f]chromen-3-ones (4a-e) have been evaluated for analgesic, antibacterial and antiviral activities. Analgesic activity was carried out using acetic acid-induced writhing method in Swiss albino male mice. The antibacterial activity was performed against Gram-positive and Gram-negative clinical strains by agar well diffusion method. The in vitro antiviral activity was carried out against camelpox and buffalopox viruses. The analgesic activity exhibited by the compounds 4a, 4c and 4d were found to be more significant compared to the standard. The bacterial activity was determined by the inhibition of growth of the organism by the drugs at different concentrations. All the compounds showed significant activity when compared with the drug ciprofloxacin. The in vitro antiviral activity of the compound 4b tested against camelpox and buffalopox viruses revealed no activity when tested at concentrations of 250 µg. The compound 4b did not alter the titres of both the viruses and the titres remain, respectively, 10(6.5) TCID50 and 10(6.74) TCID50 per ml for camelpox vaccine virus and buffalopox vaccine virus. However, the compounds 4a-e showed significant analgesic and antibacterial activities.

Outbreak of human buffalopox infection.

An outbreak of febrile illness with vesicular lesions on hands and forearms of six persons was investigated. There was simultaneous outbreak of similar illness in buffaloes that were milked by these persons. Buffaloes had lesions on many parts of body including udder and teats. Manual milking without gloves exposed the persons to the infection. Investigations proved that both the outbreaks were due to buffalopox virus infection. Improved dairy practices like wearing gloves while milking might help in prevention of spread of infection to other animals and humans.