Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

BACKGROUND: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. METHODS AND RESULTS: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. CONCLUSIONS: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

Identification of polymorphisms in the MSTN and ADRB3 genes associated with growth and ultrasound carcass traits in Kajli sheep.

The aim of this investigation was to find single nucleotide polymorphisms (SNPs) in the myostatin (MSTN) and the beta-3 adrenergic receptor (ADRB3) genes associated with growth and ultrasound carcass traits in Kajli sheep. The five growth traits were birth weight (BWT-EBV), 120-day weight (120DWT-EBV), 180-day weight (180DWT-EBV), 270-day weight (270DWT-EBV), and 365-day weight (365DWT-EBV). The three ultrasound carcass traits were width (WLD) and depth of longissimus dorsi (DLD) and back fat thickness (BFT). The analysis of the MSTN sequence revealed one non-synonymous substitution (c.197T > A) in exon 1, one single nucleotide substitution (c.373 + 18G > T) in intron 1, and one synonymous substitution (c.861T > A) in exon 3. However, there were four single nucleotide synonymous substitutions (c.130C > T, c.294C > G, c.579G > T, and c.654C > G) in exon 1 of the ADRB3 gene. All the SNPs in the MSTN gene, except for c.373 + 18G > T, were in Hardy-Weinberg Equilibrium (HWE). Conversely, none of the SNPs found in ADRB3 were in HWE. Two of the MSTN SNPs (c.197T > A and c.373 + 18G > T) had significant associations with all evaluated growth and ultrasound carcass traits. The SNPs c.130C > T and c.294C > G in ADRB3 were significantly associated with 180DWT-EBV. Collectively, these findings indicate that several SNPs in the studied genes were significantly related to growth and carcass traits in Kajli sheep.

CRISPR-CAS Replacing Antiviral Drugs against HIV: An Update.

Various antiretroviral drugs do not kill or cure the human immunodeficiency virus (HIV) but do prevent the replication of the virus. The combination of antiretroviral drugs is known as highly active antiretroviral therapy (HAART). Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attacks on this proviral DNA. The biggest achievement might be the complete elimination of HIV from infected cells. A study revealed that the tail injection, in transgenic mice and rats having HIV-1 genome, of an adenoassociated virus (AAV) vector expressing a short version of the Cas9 endonuclease (saCas9) and the gRNAs resulted in the cleavage of integrated HIV-1 DNA and excision of a DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, kidney, and circulating lymphocytes. HIV-1 has capacity to escape the attack on its genome from most of inhibitors. Thus, to achieve successful antiretroviral treatment, combinations of several antiviral therapies have been applied that are based on two important facts. The first is that multiple drugs lead to synergistic or additive inhibition, and the second is that the combinational therapy increases chances of drug resistance. The success that has been achieved with the help of the genetically engineered tool CRISPR is that dCas9 protein alone can efficiently silence viral gene expression in bacteria with sgRNA. All the reported investigations have indicated that CRISPR/Cas9 can be used as immune machinery into human cells in the form of novel antivirus tools.

Crimean-Congo Hemorrhagic Fever (CCHF) in Pakistan: The "Bell" is Ringing Silently.

Pakistan is being hit by communicable and noncommunicable diseases over time. Among these, tickborne viral disease, Crimean-Congo hemorrhagic fever (CCHF) is one of the most fatal infections. Rapid climate change aroused by industrial, occupational, and agricultural activities to support ever-growing human population has been considered the single most causative basis for emergence or re-emergence of CCHF in Pakistan, where it has biannual peaks between the months of March-May and August-October. Many factors, including poor sanitation at farms, villages, and cities, unhygienic transportation and slaughter of animals at numerous sites within a city, inefficient tick-control programs, post-slaughter piles of animal remains other than meat, nomadic lifestyle, and lack of trained animal and human healthcare staff, are contributing to the spread of CCHF. Pakistan has confirmed cases of CCHF in almost every province: Sindh (Karachi), Punjab (Faisalabad, Multan, and Rawalpindi), Balochistan (Quetta) and Khyber Pakhtunkhwa (Peshawar). The root cause behind the spread of CCHF in Pakistan seems to be the absence of an effective disease surveillance system in the human as well as the animal populations. Most of the time, CCHF cases are not diagnosed, and if they are diagnosed they are not reported. If these cases are reported, there are not enough effective measures by the relevant provincial and district authorities. There is a need to educate the general public, farmers, and healthcare workers about the causes, transmission, and dangers of CCHF. An immediate plan for the implementation of a surveillance system, standard preventive measures, early detection, proper treatment, and timely response is urgently needed. Without such a plan, the accumulation of factors responsible for the sudden outbreak of CCHF may pose a serious threat to humans and animals in different geographical regions of the country.

Deadly outbreak of chickenpox at district Faisalabad, Pakistan: possible causes, and preventive way forward.

This article details our recommendations for the deadly outbreak of chickenpox to consider the additional referral of the absence of a monitoring system of prevention and control along with poor vaccination system for children in low-resource settings. The recent spread of chickenpox outbreak in Pakistan has claimed dozens of lives. The deaths in this current outbreak in quick successions are beyond understanding. Re-emergence of chickenpox in the area has raised many questions. Keeping in view the spread of chickenpox mainly in Faisalabad and its international reputation in trading, chickenpox breakout needs international attention to control its spread. It should be taken as an eye opener for the Government of Pakistan and government should develop and implement Centralized Infectious Disease Reporting Information Management System that will help to narrow down the pathogens as far as the epidemics are concerned and also for early preventive and countermeasure response.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Resistance Patterns of Frequently Applied Antimicrobials and Occurrence of Antibiotic Resistance Genes in Edwardsiella tarda Detected in Edwardsiellosis-Infected Tilapia Species of Fish Farms of Punjab in Pakistan.

Edwardsiella tarda is one of the most significant fish pathogens, causes edwardsiellosis in a variety of freshwater fish species, and its antibiotic resistance against multiple drugs has made it a health risk worldwide. In this study, we aimed to investigate the antibiotic resistance (ABR) genes of E. tarda and establish its antibiotic susceptibility. Thus, 540 fish (299 Oreochromis niloticus, 138 O.mossambicus, and 103 O. aureus) were collected randomly from twelve fish farms in three districts of Punjab in Pakistan. E. tarda was recovered from 147 fish showing symptoms of exophthalmia, hemorrhages, skin depigmentation, ascites, and bacteria-filled nodules in enlarged liver and kidney. Antimicrobial susceptibility testing proved chloramphenicol, ciprofloxacin, and streptomycin effective, but amoxicillin, erythromycin, and flumequine ineffective in controlling edwardsiellosis. Maximum occurrence of qnrA, blaTEM, and sul3 genes of E. tarda was detected in 45% in the liver, 58%, and 42% respectively in the intestine; 46.5%, 67.2%, and 55.9% respectively in O. niloticus; 24%, 36%, and 23% respectively in summer with respect to fish organs, species, and season, respectively. Motility, H2S, indole, methyl red, and glucose tests gave positive results. Overall, E. tarda infected 27.2% of fish, which ultimately caused 7.69% mortality. The Chi-squared test of independence showed a significant difference in the occurrence of ABR genes of E. tarda with respect to sampling sites. In conclusion, the misuse of antibacterial agents has led to the emergence of ABR genes in E. tarda, which in association with high temperatures cause multiple abnormalities in infected fish and ultimately resulting in massive mortality.

Gold nanoparticles capped with L-glycine, L-cystine, and L-tyrosine: toxicity profiling and antioxidant potential.

Biomolecules-based surface modifications of nanomaterials may yield effective and biocompatible nanoconjugates. This study was designed to evaluate gold nanoconjugates (AuNCs) for their altered antioxidant potential. Gold nanoparticles (AuNPs) and their conjugates gave SPR peaks in the ranges of 512-525 nm, with red or blueshift for different conjugates. Cys-AuNCs demonstrated enhanced (p < 0.05) and Gly-AuNCs (p > 0.05) displayed reduced DPPH activity. Gly-AuNCs and Tyr-AuNCs displayed enhanced ferric-reducing power and hydrogen peroxide scavenging activity, respectively. Cadmium-intoxicated mice were exposed to gold nanomaterials, and the level of various endogenous parameters, i.e., CAT, GST, SOD, GSH, and MTs, was evaluated. GSH and MTs in liver tissues of the cadmium-exposed group (G2) were elevated (p < 0.05), while other groups showed nonsignificance deviations than the control group. It is concluded that these nanoconjugates might provide effective nanomaterials for biomedical applications. However, more detailed studies for their safety profiling are needed before their practical applications.

Drosophila Ectoderm-expressed 4 modulates JAK/STAT pathway and protects flies against Drosophila C virus infection.

Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. In this study, we show that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM. Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, our study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway.

Vaccine Adjuvants: Selection Criteria, Mechanism of Action Associated with Immune Responses and Future Directions.

The most effective method to minimize the prevalence of infectious diseases is vaccination. Vaccines enhance immunity and provide protection against different kinds of infections. Subunit vaccines are safe and less toxic, but due to their lower immunogenicity, they need adjuvants to boost the immune system. Adjuvants are small particles/molecules integrated into a vaccine to enhance the immunogenic feedback of antigens. They play a significant role to enhance the potency and efficiency of vaccines. There are several types of adjuvants with different mechanisms of action; therefore, improved knowledge of their immunogenicity will help develop a new generation of adjuvants. Many trials have been designed using different kinds of vaccine adjuvants to examine their safety and efficacy, but in practice, only a few have entered in animal and human clinical trials. However, for the development of safe and effective vaccines, it is important to have adequate knowledge of the side effects and toxicity of different adjuvants. The current review discussed the adjuvants which are available for producing modern vaccines as well as some new classes of adjuvants in clinical trials.

Molecular evolution, virology and spatial distribution of HCV genotypes in Pakistan: A meta-analysis.

Background: Hepatitis C, caused by the Hepatitis C Virus (HCV), is the second most common form of viral hepatitis. The geographical distribution of HCV genotypes can be quite complex, making it challenging to ascertain the most prevalent genotype in a specific area. Methods: To address this, a review was conducted to determine the prevalence of HCV genotypes across various provinces and as a whole in Pakistan. The scientific literature regarding the prevalence, distribution, genotyping, and epidemiology of HCV was gathered from published articles spanning the years 1996-2020. Results: Genotype 1 accounted for 5.1% of the patients, with its predominant subtype being 1a at 4.38%. The frequencies of its other subtypes, 1b and 1c, were observed to be 1.0% and 0.31% respectively. Genotype 2 had a frequency of 2.66%, with the most widely distributed subtype being 2a at 2.11% of the patients. Its other subtypes, 2b and 2c, had frequencies of 0.17% and 0.36% respectively. The most prevalent genotype among all isolates was 3 (65.35%), with the most frequent subtype being 3a (55.15%), followed by 3b (7.18%). The prevalence of genotypes 4, 5, and 6 were scarce in Pakistan, with frequencies of 0.97%, 0.08%, and 0.32% respectively. The prevalence of untypeable and mixed genotypes was 21.34% and 3.53% respectively. Estimating genotypes proves to be a productive method in assisting with the duration and selection of antiviral treatment. Different HCV genotypes can exhibit variations in their response to specific antiviral treatments. Different genotypes may have distinct natural histories, including variations in disease progression and severity. Some genotypes may lead to more rapid liver damage, while others progress more slowly. Conclusions: This information can guide screening and testing strategies, helping to identify individuals at higher risk of developing severe complications. Studying the distribution of HCV genotypes in a population can provide valuable insights into the transmission dynamics of the virus.