Alpha toxin production potential and antibiotic resistance patterns of clostridium perfringens isolates from meat samples.

Objective: This research aimed to analyze the prevalence, molecular characteristics, toxinotyping, alpha toxin production potential, and antibiotic resistance pattern of Clostridium perfringens (C. perfringens) isolates in meat samples collected from various sources. Methods: Sixty meat samples were screened for alpha toxin using Enzyme-Linked Immunosorbent Assay (ELISA), revealing a positivity rate of 13.3%, predominantly in raw poultry meat. Subsequent culturing on Perfringens agar identified nine samples harboring characteristic C. perfringens colonies, primarily isolated from raw poultry meat. Molecular confirmation through 16S rRNA gene amplification and sequencing authenticated twelve isolates as C. perfringens, with nine strains exhibiting genetic resemblance to locally isolated strains. Toxinotyping assays targeting alpha toxin-specific genes confirmed all nine isolates as type A C. perfringens, with no detection of beta or epsilon toxin genes. Hemolytic assays demonstrated varying alpha toxin production potentials among isolates, with accession number OQ721004.1 displaying the highest production capacity. Moreover, antibiotic resistance profiling revealed multi-drug resistance patterns among the isolates. Results: The study identified distinct clusters within C. perfringens strains, indicating variations. Phylogenetic analysis delineated genetic relatedness among strains, elucidating potential evolutionary paths and divergences. Conclusion: The findings underscore the need for robust surveillance and control measures to mitigate the risk of C. perfringens contamination in meat products, particularly in raw poultry meat. Enhanced monitoring and prudent antimicrobial stewardship practices are warranted in both veterinary and clinical settings to address the observed antibiotic resistance profiles and prevent foodborne outbreaks.

Evaluation of genotoxic effect via expression of DNA damage responsive gene induced by ivermectin on MDBK cell line.

Ivermectin (IVM) is an anti-parasitic drug which is used for treating parasitic infestations. It has been used in humans for treating intestinal strongyloidiasis and onchocerciasis however, currently researchers are investigating its potential for treating coronavirus SARS-CoV-2. Due to its broad-spectrum activities, IVM is being used excessively in animals which has generated an interest for researchers to investigate its toxic effects. Cytotoxic and genotoxic effects have been reported in animals due to excessive usage of IVM. Therefore, this study aims to evaluate the cytotoxic and genotoxic effects of IVM on the Madin-Darby-Bovine-Kidney (MDBK) cell line by examining the expression of a DNA damage-responsive gene (OGG1). Cytotoxicity of IVM was tested using an assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereas the genotoxicity was evaluated using comet assay along with micronucleus assay. Moreover, the gene expression of DNA damage response gene (OGG1) was measured by qRT-PCR, after extraction of RNA from the MDBK cell line using the TRIzol method and its conversion to cDNA by reverse-transcriptase PCR. During the experiment, cell viability percentage was measured at different doses of IVM i.e., 25%, 50%, 75%, along with LC50/2, LC50 and LC50*2. It was observed that the gene expression of OGG1 increased as the concentration of IVM increased. It was concluded that IVM has both cytotoxic and genotoxic effects on the MDBK cell line. Furthermore, it is recommended that studies related to the toxic effects of IVM at molecular level and on other model organisms should be conducted to combat its hazardous effects.

The immunogenicity and efficacy of acommercially available Infectious Bovine Rhinotracheitis (IBR) virus vaccine against a Pakistani field IBR strain.

Infectious bovine rhinotracheitis (IBR) is a highly communicable disease of cattle and wild ruminants that is caused by Bovine alphaherpesvirus 1 (BoHV­1). For IBR control, several developed countries have adopted the immunization and eradication programs focusing on IBR­positive animals. In Pakistan, livestock producers are importing commercially available vaccine of BoHV­1, but no studies on the efficacy of these commercial vaccines against local isolates are available. Therefore, the present study was aimed to evaluate the efficacy of a commercially available vaccine of BoHV­1 against local field isolates of virus. The rabbit model was used and the vaccine was evaluated for immunogenicity and protection after challenge with a highly virulent strain of a field virus. The immune response was measured by virus neutralization titers (VNT). This vaccine induced a humoral response in rabbits but that was not sufficient to completely protect the vaccinated animals against the wild­type BoHV­1 strain challenge. While a low virus titer compared to control rabbits was observed in the vaccinated rabbits (p<0.05), there was no sterilizing immunity or freedom from infection. However, complete freedom from disease, for example, the absence of pyrexia was noticed in the vaccinated group. In conclusion, the present study demonstrated that imported vaccine stock provoked only a partial protection against indigenous isolated of BoHV­1. However, tests performed on rabbits are preliminary, as only those performed on the source species can determine more reliable results.

Design and Assessment of a Novel In Silico Approach for Developing a Next-Generation Multi-Epitope Universal Vaccine Targeting Coronaviruses.

In the past two decades, there have been three coronavirus outbreaks that have caused significant economic and health crises. Biologists predict that more coronaviruses may emerge in the near future. Therefore, it is crucial to develop preventive vaccines that can effectively combat multiple coronaviruses. In this study, we employed computational approaches to analyze genetically related coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, focusing on the spike glycoprotein as a potential vaccine candidate. By predicting common epitopes, we identified the top epitopes and combined them to create a multi-epitope candidate vaccine. The overall quality of the candidate vaccine was validated through in silico analyses, confirming its antigenicity, immunogenicity, and stability. In silico docking and simulation studies suggested a stable interaction between the multi-epitope candidate vaccine and human toll-like receptor 2 (TLR2). In silico codon optimization and cloning were used to further explore the successful expression of the designed candidate vaccine in a prokaryotic expression system. Based on computational analysis, the designed candidate vaccine was found to be stable and non-allergenic in the human body. The efficiency of the multi-epitope vaccine in triggering effective cellular and humoral immune responses was assessed through immune stimulation, demonstrating that the designed candidate vaccine can elicit specific immune responses against multiple coronaviruses. Therefore, it holds promise as a potential candidate vaccine against existing and future coronaviruses.

Molecular Characterization of UL50 (dUTPase) Gene of Bovine Herpes Virus 1.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells.

Lack of antiviral activity of ivermectin against foot-and-mouth disease virus serotype O in BALB/c mice.

Ivermectin is an FDA approved drug and showed in vitro antiviral activity against different serotypes of Foot-and-mouth disease virus (FMDV). We here assessed the effect of ivermectin in 12 day old female BALB/c mice infected with 50LD50 FMDV serotype O intraperitoneally. Initially FMDV was adopted on 3-day old BALB/c mice by blind passages. After successful adaptation of virus mice showed hind limb paralysis. Mice were divided in 6 different groups and each group has 6 mice. Ivermectin was given at clinically prescribed dose of 500 µg/kg subcutaneously at different time interval. Ivermectin was given at 0 h post infection (hpi) and 12 hpi. Moreover we compared commercially available ivermectin with purified ivermectin preparation in sterilized DMSO. Viral load was evaluated through RT-qPCR and ELISA in different groups. Results showed that positive control and negative control has CT-value 26.28 and 38 respectively. Treated groups at 0hpi, 12hpi, purified ivermectin and pre-post treatment group has CT values 24.89, 29.44, 27.26 and 26.69 respectively that showed there was no significant reduction in virus load in treated groups as compare to positive control. In histopathology of lung tissue perialveolar capillaries were congested and alveoli were altelactic. Some emphysema was seen in alveoli and mild thickening in the alveolar wall was observed. In the alveolar epithelium mononuclear cells infiltration was seen. There was discoloration haemorrhages and enlargement of heart. Degeneration, fragmentation and loss of sarcoplasm were seen in the cardiac muscle fibers. Above results showed that ivermectin did not lessen lung and heart viral load. This study contributes that ivermectin does not have a significant antiviral effect when used in mice against FMDV serotype O, according to a growing body of research.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

An In silico study of derivative of Newcastle disease virus epitopes based vaccine against Hemagglutunin neuraminidase protein.

The causative agent of Newcastle disease (ND) is Newcastle disease virus. It belongs to avian species of Orthoavulavirus, Avulavirinae subfamily and if left untreated it may cause epidemic in poultry. Many vaccines have been made against Newcastle disease based on inactivated and attenuated viruses but become useless due to the genetic changes in the virus. We have recently reported epitope based vaccine by using immunoinformatics approaches. The vaccine was previously constructed against Hemagglutunin neuraminidase protein of Newcastle disease virus. Here we extended our work to develop several chimera of the proposed vaccine to design a new multi-epitope vaccine by shuffling the cytotoxic T lymphocytes (CTL) segments of the vaccine. Total 5040 constructs have been analyzed by shuffling 7 CTL epitopes. Highest antigenic multi-epitope construct was selected for the further study. Our new multi-epitope vaccine (MEV) construct contains 259 amino acids and is immunogenic, more antigenic and non-allergen. The refinement of the structure of MEV construct was performed. Molecular docking analyses showed its maximum binding with avian Toll-like 4 receptor. Subsequently, immune simulations showed its predicted ability to induce the host primary and secondary responses. Study suggests that our new multi-epitope vaccine chimera is more effective and stable protein against Newcastle disease virus strains in Pakistan. However, further studies are required to validate the vaccine through In vitro and In vivo studies.

Renessans Helps in Early Clearance of SARS-CoV-2: In-Vivo Activity of the Iodine Complex in Rhesus macaque.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

Prediction and analysis of multi epitope based vaccine against Newcastle disease virus based on haemagglutinin neuraminidase protein.

Newcastle disease virus (NDV), an avian orthoavulavirus, is a causative agent of Newcastle disease named (NDV), and can cause even the epidemics when disease is not treated. Previously several vaccines based on attenuated and inactivated viruses have been reported which are rendered useless with the passage of time due to versatile changes in viral genome. Therefore, we aimed to develop an effective multi-epitope vaccine against the haemagglutinin neuraminidase (HN) protein of 26 NDV strains from Pakistan through a modern immunoinformatic approaches. As a result, a vaccine chimaera was constructed by combining T-cell and B-cell epitopes with the appropriate linkers and adjuvant. The designed vaccine was highly immunogenic, non-allergen and antigenic; therefore, the potential 3D-structureof multi epitope vaccine was constructed, refined and validated. A molecular docking study of a multiepitope vaccine candidate with the chicken Toll-like receptor-4 indicated successful binding. An In silico immunological simulation was used to evaluate the candidate vaccine's ability to elicit an effective immune response. According to the computational studies, the proposed multiepitope vaccine is physically stable and may induce immune responses whichsuggested it a strong candidate against 26 Newcastle disease virus strains from Pakistan.

A Cross-Sectional Survey of Knowledge, Attitude, and Practices of University Students in Pakistan Regarding COVID-19.

The COVID-19 pandemic is striking the world with serious public health and socioeconomic complications. The pandemic has influenced all forms of daily life, including educational institutions. Therefore, this cross-sectional survey was conducted to understand knowledge, attitudes, and practices related to COVID-19 among the students of the University of Veterinary and Animal Sciences, Lahore. The data was collected using an online self-directed questionnaire. The survey form includes six items about sociodemographic characteristics, 14 knowledge-based questions, seven questions on attitude, and eight questions on practices. The sample number was calculated using the Raosoft sample size calculator. A total number of 3,854 students, including 1,823 men and 2,031 women, were engaged in this survey, having student representation from all the provinces in the country. The data were analyzed using a chi-square test. A total of 97% of the students knew that the etiological agent of COVID-19 is a virus and that it is a disease of the respiratory system (94%). Many students kept visiting their relatives during the lockdown (45%), and their relatives kept visiting them at home (59%). The responses from the students varied a lot on specific questions about the transmission of the virus. Women tended to have less information regarding precautionary travel measures (p < 0.01), but supplemental knowledge of prevention of disease transmission from positive patients (p < 0.01). Conclusively, the majority of the university students surveyed had imperative knowledge, a good attitude, and active practice in response to the COVID-19 outbreak. Moreover, the KAP scores have varied by demography, gender, and the number of family members. Therefore, continuous awareness of preventative behaviors should be disseminated regularly in emergencies.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

Diphenhydramine and levofloxacin combination therapy against antimicrobial resistance in respiratory tract infections.

Background: Antibiotics are in use since decades to treat various infections caused by Gram-positive and Gram-negative bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. Diphenhydramine, an H1 receptor blocker possesses a weak antibiotic action but when combined with other antibiotics may potentiate their antibacterial activity. Materials & methods: This study investigated in vitro antibacterial activity of diphenhydramine when used alone and in combination with levofloxacin against methicillin-resistant S. aureus and P. aeruginosa. Results: The combined antibacterial effect of the drugs against bacteria showed a fractional inhibitory concentration index of ≤0.5, in other words, synergism. No cytotoxicity was observed as percentage cell viability was >50%. Conclusion: The combination of diphenhydramine and levofloxacin exerted antibacterial activity, and was not found to be cytotoxic when given in combination against P. aeruginosa and S. aureus.

Antiviral potential of ivermectin against foot-and-mouth disease virus, serotype O, A and Asia-1.

Each year, foot-and-mouth disease leads to enormous economic losses to the livestock industry. Currently, the killed whole virus is widely using to control FMD. However, vaccination is constrained by lack of or incomplete protection. Therefore, along with vaccination, we need to find the antivirals against FMD. This study was conducted to investigate the antiviral potential of ivermectin against multiple serotypes of FMDV. Initially, an MTT assay was performed on the BHK-21 cell line to determine assay ivermectin cytotoxicity. Viral inhibition assays using the non-cytotoxic concentration of ivermectin were performed to check the antiviral potential of ivermectin on different stages of virus replication. At 2.5 µM and 5 µM concentrations of ivermectin, the virus titer was reduced significantly (p < 0.001) by two to three log in all three strains of viruses at both non-toxic concentrations (2.5 and 5 µM). The virus titer in strain O control was 106.0 TCID50/0.1 mL and was reduced to 104.1 TCID50/0.1 mL at a concentration of 2.5 µM and 103.10 TCID50/0.1 mL at 5 µM concentration. In the case of strain Asia-1, the virus titer was reduced to 103.8 TCID50/0.1 mL at 2.5 µM and 103.01TCID50/0.1 mL at 5 µM concentration. The titer of strain A was reduced from 105.8 TCID50/0.1 mL to 103.9 TCID50/0.1 mL at 2.5 µM concentration and 103.1 TCID50/0.1 mL at 5 µM concentration. Moreover, the virus titer was reduced more at the replication stage as compared to attachment and entry stages. This study showed the in vitro anti-FMDV potential of ivermectin for the first time and predicted its potential use against FMDV infections.

Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes with Viral Replication.

Bovine herpesvirus1 (BoHV-1) is a major bovine pathogen. Despite several vaccines being available to prevent viral infection, outbreaks are frequent and cause important economic consequences worldwide. The development of new antiviral drugs is therefore highly desirable. In this context, viral genome replication represents a potential target for therapeutic intervention. BoHV-1 genome is a dsDNA molecule whose replication takes place in the nuclei of infected cells and is mediated by a viral encoded DNA polymerase holoenzyme. Here, we studied the physical interaction and subcellular localization of BoHV-1 DNA polymerase subunits in cells for the first time. By means of co-immunoprecipitation and confocal laser scanning microscopy (CLSM) experiments, we could show that the processivity factor of the DNA polymerase pUL42 is capable of being autonomously transported into the nucleus, whereas the catalytic subunit pUL30 is not. Accordingly, a putative classic NLS (cNLS) was identified on pUL42 but not on pUL30. Importantly, both proteins could interact in the absence of other viral proteins and their co-expression resulted in accumulation of UL30 to the cell nucleus. Treatment of cells with Ivermectin, an anti-parasitic drug which has been recently identified as an inhibitor of importin α/ß-dependent nuclear transport, reduced UL42 nuclear import and specifically reduced BoHV-1 replication in a dose-dependent manner, while virus attachment and entry into cells were not affected. Therefore, this study provides a new option of antiviral therapy for BoHV-1 infection with Ivermectin.

In silico analysis of four structural proteins of aphthovirus serotypes revealed significant B and T cell epitopes.

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.

Role of Rab GTPases in HSV-1 infection: Molecular understanding of viral maturation and egress.

Most enveloped viruses exploit complex cellular pathways for assembly and egress from the host cell, and the large DNA virus Herpes simplex virus 1 (HSV-1) makes no exception, hijacking several cellular transport pathways for its glycoprotein trafficking and maturation, as well as for viral morphogenesis and egress according to the envelopment, de-envelopment and re-envelopment model. Importantly Rab GTPases, widely distributed master regulators of intracellular membrane trafficking pathways, have recently being tightly implicated in such process. Indeed, siRNA-mediated genetic ablation of specific Rab proteins differently affected HSV-1 production, suggesting a complex role of different Rab proteins in HSV-1 life cycle. In this review, we discuss how different Rabs can regulate HSV-1 assembly/egress and the potential therapeutic applications of such findings for the management of HSV-1 infections.

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses.

Bovine herpesvirus 1 tegument protein UL21 plays critical roles in viral secondary envelopment and cell-to-cell spreading.

Bovine herpesvirus 1 (BoHV-1) UL21 is a tegument protein thought to be indispensable for efficient viral growth but its precise function in BoHV-1 is currently unknown. To determine the function of UL21 in BoHV-1 replication, we constructed a mutant virus bearing a UL21 deletion (vBoHV-1-∆UL21) and its revertant virus, vBoHV-1-∆UL21R, in which the UL21 gene was restored using a bacterial artificial chromosome system. The replication of vBoHV-1-∆UL21 was 1,000-fold lower and its plaque size was 85% smaller than those of the wild-type virus (BoHV-1). An ultrastructural analysis showed that deletion of UL21 led to an un-enveloped capsid accumulation in the cytoplasm, whereas nucleocapsid egress was not impaired, suggesting that UL21 is critical for secondary envelopment in BoHV-1. Co-immunoprecipitation assays revealed that HA-tagged UL21 pulled down UL16, suggesting that these two proteins form a complex, and this was further confirmed by a co-immunofluorescence assay. Taken together, these data provide evidence that UL21 plays critical roles in BoHV-1 secondary envelopment, and UL16 is likely to be involved in these activities.

A bovine herpesvirus 1 pUL51 deletion mutant shows impaired viral growth in vitro and reduced virulence in rabbits.

Bovine herpesvirus 1 (BoHV-1) UL51 protein (pUL51) is a tegument protein of BoHV-1 whose function is currently unknown. Here, we aimed to illustrate the specific role of pUL51 in virion morphogenesis and its importance in BoHV-1 virulence. To do so, we constructed a BoHV-1 bacterial artificial chromosome (BAC). We used recombinant BAC and transgenic techniques to delete a major part of the UL51 open reading frame. Deletion of pUL51 resulted in severe viral growth defects, as evidenced by lower single and multi-step growth kinetics, reduced plaque size, and the accumulation of non-enveloped capsids in the cytoplasm of infected cells. Using tagged BoHV-1 recombinant viruses, it was determined that the pUL51 protein completely co-localized with the cis-Golgi marker protein GM-130. Taken altogether, pUL51 was demonstrated to play a critical role in BoHV-1 growth and it is involved in viral maturation and egress. Moreover, an in vivo analysis showed that the pUL51 mutant exhibited reduced virulence in rabbits, with no clinical signs, no nasal shedding of the virus, and no detectable serum neutralizing antibodies. Therefore, we conclude that the BoHV-1 pUL51 is indispensable for efficient viral growth in vitro and is essential for virulence in vivo.