Laboratory Methods for the Diagnosis of SARS-Cov-2

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

Immune Evaluation of Recombinant Lactobacillus plantarum With Surface Display of HA1-DCpep in Mice

Avian influenza viruses can be efficiently transmitted through mucous membranes, and conventional vaccines are not effective in protecting against mucosal infection by influenza viruses. To induce multiple immune responses in an organism, we constructed a recombinant Lactobacillus plantarum expressing the influenza virus antigen HA1 with the adjuvant dendritic cell-targeting peptide (DCpep). The recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep were used to immunize mice via oral administration, and the humoral, cellular and mucosal immune responses were evaluated. In addition, the serum levels of specific antibodies and hemagglutination inhibition (HI) levels were also measured. Our results showed that recombinant L. plantarum activated dendritic cells in Peyer’s patches (PPs), increased the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes (MLNs), and affected the ability of CD4+ and CD8+ cells to proliferate in the spleen and MLNs. Additionally, recombinant L. plantarum increased the number of B220+IgA+ cells in PPs and the level of IgA in the lungs and different intestinal segments. In addition, specific IgG, IgG1 and IgG2a antibodies were induced at high levels in the mice serum, specific IgA antibodies were induced at high levels in the mice feces, and HI potency was significantly increased. Thus, the recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep have potential as vaccine candidates for avian influenza virus.

Smart investment of virus RNA testing resources to enhance Covid-19 mitigation

A variety of mitigation strategies have been employed against the Covid-19 pandemic. Social distancing is still one of the main methods to reduce spread, but it entails a high toll on personal freedom and economic life. Alternative mitigation strategies that do not come with the same problems but are effective at preventing disease spread are therefore needed. Repetitive mass-testing using PCR assays for viral RNA has been suggested, but as a stand-alone strategy this would be prohibitively resource intensive. Here, we suggest a strategy that aims at targeting the limited resources available for viral RNA testing to subgroups that are more likely than the average population to yield a positive test result. Importantly, these pre-selected subgroups include symptom-free people. By testing everyone in these subgroups, in addition to symptomatic cases, large fractions of pre- and asymptomatic people can be identified, which is only possible by testing-based mitigation. We call this strategy smart testing (ST). In principle, pre-selected subgroups can be found in different ways, but for the purpose of this study we analyze a pre-selection procedure based on cheap and fast virus antigen tests. We quantify the potential reduction of the epidemic reproduction number by such a two-stage ST strategy. In addition to a scenario where such a strategy is available to the whole population, we analyze local applications, e.g. in a country, company, or school, where the tested subgroups are also in exchange with the untested population. Our results suggest that a two-stage ST strategy can be effective to curb pandemic spread, at costs that are clearly outweighed by the economic benefit. It is technically and logistically feasible to employ such a strategy, and our model predicts that it is even effective when applied only within local groups. We therefore recommend adding two-stage ST to the portfolio of available mitigation strategies, which allow easing social distancing measures without compromising public health.

Immunogenic properties of the preparation containing the Chikungunya virus antigen inactivated by β-propiolactone

Introduction. Cases of Chikungunya fever have been reported in more than 100 countries in Europe, Oceania, Africa, Asia, the Caribbean, and America. The musculoskeletal disorders typical for Chikungunya fever can last from several months to a year and even lead to disability. The infection is believed to provide lifelong immunity. This factor and the lack of specific therapy make vaccination the most promising method for preventing Chikungunya fever.Materials and methods. The purified inactivated preparation with the different doses of the CHIKV antigen was injected intramuscularly to BALB/c mice twice with an interval of 14 days. Indicators of humoral and cellular immunity were assessed in dynamics in ELISA, the neutralization test and proliferation test of splenocytes. Results. The purified preparation containing the CHIKV antigen inactivated by beta-propiolactone had pronounced immunogenic properties. The most prominent immune response in ELISA and neutralization test was registered for a dose of 40 μg. Stimulation with the specific CHIKV antigen caused a pronounced proliferation of animals' splenocytes. The peak values of specific humoral and cellular immunity parameters were registered 14 days after the second injection.Discussion. The purified preparation containing the CHIKV antigen inactivated by beta-propiolactone had demonstrated the sufficient immunogenic properties. The immunizing dose of 40 μg CHIKV selected as a result of the studies caused in BALB/c mice the development of the humoral immunity characterized by the specific IgG with neutralizing activity, and the specific cell immunity characterized by the animals' splenocytes proliferation after stimulation with CHIKV antigen.Conclusion. The purified β-PL inactivated preparation of the CHIKV antigen at a dose of 40 μg to demonstrated pronounced immunogenicity in BALB/c mice after two-dose immunization. The developed preparation can be considered as promising for the prevention of Chikungunya fever using the dose and scheme tested in this study.

RDT BASED DETECTION OF HEPATITIS B SURFACE ANTIGEN (HBSAG) AMONG BLOOD DONORS AT SPECIALIST HOSPITAL, SOKOTO

Hepatitis B infection is a major global health problem and a potentially life-threatening infection that attacks liver cells caused by Hepatitis B virus and it is characterised by liver cirrhosis, liver scarring, liver failure or cancer of the liver as the disease progresses. This study was carried out to determine theRDT based detection of hepatitis B surface antigen (HbsAg) among blood donors at specialist hospital, Sokoto, Sokoto State Nigeria. Two hundred (200) centrifuged sera from blood donors who have given prior verbal consent to participate in the study were screened for HBsAg using rapid diagnostic test (RDT). The result obtained shows that, 51 out of the 200 blood donors which represented (25.5%) were reactive while 149 were non-reactive. With Chi-square analysis only blood donors that are none previous blood donors showed a significant association with HBV infection, (p-value 0.05). This study however, indicates that, hepatitis B infection occurs in one out of every four blood donors. It also observed that the virus antigen is highly endemic among blood donor and these blood donors can serve as potential reservoir for the transmission of the virus to susceptible host individuals in the study area.

ANTIBODY DETECTION OF NEWCASTLE DISEASE VIRUS IN SOME SPECIES OF POULTRY IN MAIDUGURI NIGERIA.

This study was conducted to determine the presence of Newcastle disease (ND) virus in Poultry in Maiduguri. A total of 100 blood sample were collected for laboratory examination. The HAand HI tests following the procedures described in the OIE reference manual. ND viral antibody was detected in sera. The prevalence of 66 (66.0%) ND among different avian species was recorded, this research further revealed that 43 (60.5%) Local chicken, 3 (100%) Broilers and 20 (76.9%) Pigeons were sero-positive for ND virus antigen. The prevalence of ND among species based on age was reported with an overall prevalence of 66 (66.0%), This research also revealed that 22 (81.4%) Young and 44 (60.0%) Female were seropositive for ND virus antigen. This result further revealed 9 (9%) Male and 57 (57%) Female were sero-positive for ND virus antigen, based on Gender distribution. Furthermore the geometric mean of Haemagglutination titre value of 76.5068 was recorded.

↵Peptide based Indirect ELISA for Detection of Antibodies against Peste des petits ruminants virus

Background: Infectious virus antigen is not recommended for disease monitoring in global Peste des petits ruminants eradication strategies. Native virus antigens are gradually being replaced with recombinant or synthetic peptide antigens. The focus of the present study is to optimize and develop peptide-based immunoassay for the detection of antibodies to PPRV N and H proteins. Methods: Epitopes of PPRV H and N proteins were selected based on prediction with bioinformatics tools and from previous studies. Two peptides each were synthesized for N and H proteins and peptide ELISA developed. The peptide ELISA’s sensitivity and specificity were tested with sera samples collected at different time intervals of vaccination (goat =73, sheep= 62) and 88 random serum samples (goat =47, sheep=41). The collected sera were screened using cELISA before proceeding to peptide ELISA. Result: In competitive ELISA, 106 goat serum samples and 96 sheep serum samples were found to be positive. Fourteen goat serum samples and seven sheep serum sample were shown to be negative. Among120 goat serum samples tested, 114 were found to be positive by peptide ELISA. Similarly, out of 103 sheep serum samples analyzed, 96 were found to be positive with peptide ELISA. The peptide ELISA based on the highly conserved and antigenic N and H epitope detected antibodies to PPRV in precise manner. This study demonstrated the effective use of synthetic peptides as an antigen in the detection of antibodies to PPRV.

Evaluation of Lesions and Viral Antigen Distribution in Domestic Pigs Inoculated Intranasally with African Swine Fever Virus Ken05/Tk1 (Genotype X)

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.