Breaking the Barrier: SARS-CoV-2 Infections in Wild and Companion Animals and Their Implications for Public Health.

The emergence of the novel coronavirus SARS-CoV-2 has led to significant interest in its potential transmission between animals and humans, especially pets. This review article summarises the literature on coronavirus infections in domestic animals, emphasising epidemiology, transmission dynamics, clinical manifestations, and public health implications. This article highlights current understandings of the relationship between infections in companion animals and humans, identifies research gaps, and suggests directions for future research. Cases of disease in cats, dogs, and other domestic animals, often occurring through close contact with infected owners, are reviewed, raising concerns about possible zoonotic and reverse zoonotic transmission. Precautions and recommendations for pet owners and healthcare workers are also discussed. The scientific evidence presented in the article highlights the need for a One Health approach that considers the health of people, animals, and the environment to combat future pandemics.

Pre-Clinical Safety and Immunogenicity Study of a Coronavirus Protein-Based Subunit Vaccine for COVID-19.

Creating an effective and safe vaccine is critical to fighting the coronavirus infection successfully. Several types of COVID-19 vaccines exist, including inactivated, live attenuated, recombinant, synthetic peptide, virus-like particle-based, DNA and mRNA-based, and sub-unit vaccines containing purified immunogenic viral proteins. However, the scale and speed at which COVID-19 is spreading demonstrate a global public demand for an effective prophylaxis that must be supplied more. The developed products promise a bright future for SARS-CoV-2 prevention; however, evidence of safety and immunogenicity is mandatory before any vaccine can be produced. In this paper, we report on the results of our work examining the safety, toxicity, immunizing dose choice, and immunogenicity of QazCoVac-P, a Kazakhstan-made sub-unit vaccine for COVID-19. First, we looked into the product's safety profile by assessing its pyrogenicity in vaccinated rabbit models and using the LAL (limulus amebocyte lysate) test. We examined the vaccine's acute and sub-chronic toxicity on BALB/c mice and rats. The vaccine did not cause clinically significant toxicity-related changes or symptoms in our toxicity experiments. Finally, we performed a double immunization of mice, ferrets, Syrian hamsters, and rhesus macaques (Macaca mulatta). We used ELISA to measure antibody titers with the maximum mean geometric titer of antibodies in the animals' blood sera totaling approximately 8 log2. The results of this and other studies warrant recommending the QazCoVac-P vaccine for clinical trials.

Coding Complete Genome Sequence of the SARS-CoV-2 Virus Strain, Variant B.1.1, Sampled from Kazakhstan.

This article describes the results of sequencing and analysis of the entire genome of the SARS-CoV-2 virus sampled in Kazakhstan in 2021. The whole-genome sequence of the strain was 29,751 bp. According to the results of phylogenetic analysis (according to the Pangolin COVID-19 database), the SARS-CoV-2/human/KAZ/B1.1/2021 strain studied here was assigned to variant B.1.1.

Near-Complete Genome Sequence of a SARS-CoV-2 Variant B.1.1.7 Virus Strain Isolated in Kazakhstan.

This research describes the genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) obtained from a patient with symptoms of coronavirus disease 2019 (COVID-19) who was infected in the Republic of Kazakhstan. Strain SARS-CoV-2/human/KAZ/Britain/2021 consists of 29,815 nucleotides and belongs to lineage B.1.1.7, according to the Pangolin COVID-19 database.

Safety and immunogenicity of the first Kazakh inactivated vaccine for COVID-19.

This article describes the results of a preclinical safety and immunogenicity study of QazCovid-in®, the first COVID-19 vaccine developed in Kazakhstan, on BALB/c mice, rats, ferrets, Syrian hamsters and rhesus macaques (Macaca mulatta). The study's safety data suggests that this immunobiological preparation can be technically considered a Class 5 nontoxic vaccine. The series of injections that were made did not produce any adverse effect or any change in the general condition of the model animals' health, while macroscopy and histology studies identified no changes in the internal organs of the BALB/c mice and rats. This study has demonstrated that a double immunization enhances the growth of antibody titers as assessed by the microneutralization assay (MNA) and the enzyme-linked immunosorbent assay (ELISA) in a pre-clinical immunogenicity test on animal models. The best GMT results were assessed in MNA and ELISA 7 days after re-vaccination; however, we noted that GMT antibody results in ELISA were lower than in MNA. A comparative GMT assessment after the first immunization and the re-immunization identified significant differences between model animal groups and a growth of GMT antibodies in all of them; also, differences between the gender groups were statistically significant. Moreover, the most marked MNA immune response to the QazCovid-in® vaccine was seen in the Syrian hamsters, while their SARS-CoV-2-specific antibody activity as assessed with ELISA was the lowest.

Efficacy and safety of an inactivated whole-virion vaccine against COVID-19, QazCovid-in®, in healthy adults: A multicentre, randomised, single-blind, placebo-controlled phase 3 clinical trial with a 6-month follow-up.

Background: Vaccination remains the primary measure to prevent the spread of the SARS-CoV-2 virus, further necessitating the use of effective licensed vaccines. Methods: From Dec 25, 2020, to July 11, 2021, we conducted a multicenter, randomised, single-blind, placebo-controlled phase 3 efficacy trial of the QazCovid-in® vaccine with a 180-day follow-up period in three clinical centres in Kazakhstan. A total of 3000 eligible participants aged 18 years or older were randomly assigned (4:1) to receive two doses of the vaccine (5 µg each, 21 days apart) or placebo administered intramuscularly. QazCovid-in® is a whole-virion formaldehyde-inactivated anti-COVID-19 vaccine, adjuvanted with aluminium hydroxide. The primary endpoint was the incidence of symptomatic cases of the SARS-CoV-2 infection confirmed by RT-PCR starting from day 14 after the first immunisation. The trial was registered with ClinicalTrials.gov NCT04691908. Findings: The QazCovid-in® vaccine was safe over the 6-month monitoring period after two intramuscular immunisations inducing only local short-lived adverse events. The concomitant diseases of participants did not affect the vaccine safety. Out of 2400 vaccinated participants, 31 were diagnosed with COVID-19; 43 COVID-19 cases were recorded in 600 placebo participants with onset of 14 days after the first dose within the 180-day observation period. Only one severe COVID-19 case was identified in a vaccine recipient with a comorbid chronic heart failure. The protective efficacy of the QazCovid-in® vaccine reached 82·0% (95% CI 71.1-88.5) within the 180-day observation period. Interpretation: Two immunisations with the inactivated QazCovid-in® vaccine achieved 82·0% (95% CI 71.1-88.5) protective efficacy against COVID-19 within a 180-day follow-up period. Funding: The work was funded by the Science Committee of the Ministry of Education and Science of Kazakhstan within the framework of the Scientific and Technical Program "Development of a vaccine against coronavirus infection COVID-19". State registration number 0.0927.

Development of the Inactivated QazCovid-in Vaccine: Protective Efficacy of the Vaccine in Syrian Hamsters.

In March 2020, the first cases of the human coronavirus disease COVID-19 were registered in Kazakhstan. We isolated the SARS-CoV-2 virus from clinical materials from some of these patients. Subsequently, a whole virion inactivated candidate vaccine, QazCovid-in, was developed based on this virus. To develop the vaccine, a virus grown in Vero cell culture was used, which was inactivated with formaldehyde, purified, concentrated, sterilized by filtration, and then adsorbed on aluminum hydroxide gel particles. The formula virus and adjuvant in buffer saline solution were used as the vaccine. The safety and protective effectiveness of the developed vaccine were studied in Syrian hamsters. The results of the studies showed the absolute safety of the candidate vaccine in the Syrian hamsters. When studying the protective effectiveness, the developed vaccine with an immunizing dose of 5 µg/dose specific antigen protected animals from a wild homologous virus at a dose of 104.5 TCID50 /mL. The candidate vaccine induced the formation of virus-neutralizing antibodies in vaccinated hamsters at titers of 3.3 ± 1.45 log2 to 7.25 ± 0.78 log2, and these antibodies were retained for 6 months (observation period) for the indicated titers. No viral replication was detected in vaccinated hamsters, protected against the development of acute pneumonia, and ensured 100% survival of the animals. Further, no replicative virus was isolated from the lungs of vaccinated animals. However, a virulent virus was isolated from the lungs of unvaccinated animals at relatively high titers, reaching 4.5 ± 0.7 log TCID50/mL. After challenge infection, 100% of unvaccinated hamsters showed clinical symptoms (stress state, passivity, tousled coat, decreased body temperature, and body weight, and the development of acute pneumonia), with 25 ± 5% dying. These findings pave the way for testing the candidate vaccine in clinical human trials.

Safety and immunogenicity of a QazCovid-in® inactivated whole-virion vaccine against COVID-19 in healthy adults: A single-centre, randomised, single-blind, placebo-controlled phase 1 and an open-label phase 2 clinical trials with a 6 months follow-up in Kazakhstan.

BACKGROUND: A new inactivated whole-virion QazCovid-in® vaccine against COVID-19 was developed from SARS-CoV-2 isolated in Kazakhstan, inactivated by formaldehyde, and adjuvanted with aluminium hydroxide. Phase 1 and 2 clinical trials aimed at assessing the vaccine's safety, immunogenicity, and the duration of immunity induced by the QazCovid-in® vaccine after one or two immunisations. METHODS: From 23.09.2020 to 19.03.2021 we performed a randomised, single-blind, placebo-controlled phase 1 clinical trial and from 18.10.2020 to 17.04.2021 an open-label phase 2 clinical trials of the QazCovid-in® vaccine with a 6 months follow-up at a single centre in Almaty, the Republic of Kazakhstan. Eligible healthy adults aged 18 years and older with no history of laboratory-confirmed SARS-CoV-2 infection were randomly assigned to the treatment groups using a computerised randomisation scheme generator. In the phase 1 clinical trial, two doses of the vaccine (5 µg each) or placebo (0·9% NaCl) were administered intramuscularly to 44 subjects aged 18-50 years, 21 days apart. In the phase 2 trial, 200 healthy participants were randomised into four equal-sized groups according to the age (18-49 or ≥50 years) and either single (day 1) or double (day 1 and 21) vaccination protocol. The primary outcomes were safety and tolerability. The secondary outcome was immunogenicity. The cellular response was measured by a whole-blood cytokine release assay (phase 1 only). The trials were registered with ClinicalTrials.gov NCT04530357. FINDINGS: The QazCovid-in® vaccine was safe and well-tolerated and induced predominantly mild adverse events; no serious or severe adverse events were recorded in both trials. In the phase 1 trial, the percentage of subjects with a fourfold increase of antibody titres (sero conversion) in MNA was 59% after one vaccine dose and amounted to 100% after two doses. Neutralizing antibody titres reached the geometric mean titre (GMT) of 100 after administration of two doses. A statistically significant increase in the levels of pro-inflammatory cytokines after vaccination indicated the Th1-biased response. On day 180, 40% of placebo-treated subjects demonstrated a statistically significant increase in the levels of antibodies measured by both ELISA and MNA, which suggests the infection with SARS-CoV-2. In the phase 2 trial, 100% of subjects aged 18-49 years seroconverted for SARS-CoV-2 on day 21 after the first dose, as indicated by MNA yielding the GMTs of 32 or 30 in the one- and two-dose groups, respectively. Amongst ≥50-year-old subjects, the number of sero conversions in the two- and one-dose groups on day 21 was 94% and 92% with the respective GMTs of 25 and 24. After the second dose, the sero conversion rate reached 100%; however, the GMT was significantly lower when compared with the corresponding value measured in subjects aged 18-49 years (83 vs 143). In both trials, specific antibodies were detected in MNA and ELISA on study day 180, but the titres dropped in comparison to day 42. The results of this study serve as the rationale for the phase 3 study. INTERPRETATION: The QazCovid-in® vaccine is safe and well-tolerated and promotes pronounced humoral immunity which lasts for at least 6 months after double intramuscular immunisation. FUNDING: The work was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan within the framework of the Scientific and Technical Program "Development of a vaccine against coronavirus infection COVID-1900 . State registration number ?.0927.

Retention of lumpy skin disease virus in Stomoxys spp (Stomoxys calcitrans, Stomoxys sitiens, Stomoxys indica) following intrathoracic inoculation, Diptera: Muscidae.

Lumpy skin disease (LSD) is an emerging disease of cattle in Kazakhstan and the means of transmission remains uncertain. In the current study, retention of Lumpy Skin Disease Virus (LSDV) by three Stomoxys species following intrathoracic inoculation was demonstrated under laboratory conditions. A virulent LSDV strain was injected into the thorax of flies to bypass the midgut barrier. The fate of the pathogen in the hemolymph of the flies was examined using PCR and virus isolation tests. LSDV was isolated from all three Stomoxys species up to 24h post inoculation while virus DNA was detectable up to 7d post inoculation.

Lumpy skin disease in Kazakhstan.

This study describes the registration of the first cases of lumpy skin disease in July 2016 in the Republic of Kazakhstan. In the rural district of Makash, Kurmangazinsky district of Atyrau region, 459 cattle fell ill and 34 died (morbidity 12.9% and mortality 0.96%). To determine the cause of the disease, samples were taken from sick and dead animals, as well as from insects and ticks. LSDV DNA was detected by PCR in all samples from dead animals and ticks (Dermacentor marginatus and Hyalomma asiaticum), in 14.29% of samples from horseflies (Tabanus bromius), and in one of the samples from two Stomoxys calcitrans flies. The reproductive LSD virus was isolated from organs of dead cattle and insects in the culture of LT and MDBK cells. The virus accumulated in cell cultures of LT and MDBK at the level of the third passage with titers in the range of 5.5-5.75 log 10 TCID50/cm3. Sequencing of the GPCR gene allowed us to identify this virus as a lumpy skin disease virus.

The combined effects of temperature and relative humidity parameters on the reproduction of Stomoxys species in a laboratory setting.

In this study, Stomoxys species (S. calcitrans, S. sitiens and S. indica) were examined to improve on the current technique for mass rearing using a method of combined incubation parameters. Moreover, the reproductive potential of immature forms at various stages of development was defined. Immature forms of stable flies were incubated according to species. There was no significant difference in the number of immature forms obtained among species incubated under the same conditions. Six incubation parameters were used in combination, at temperatures (T) of 32°C, 27°C and 22°C and relative humidity (RH) of 90% and 70% RH. The combined method resulted in a higher number of eggs hatching at 32°C and 90% humidity as well as an increase in the number of larva pupated and emergence of imago at 27°C and 70% humidity.

Mechanical Transmission of Lumpy Skin Disease Virus by Stomoxys Spp (Stomoxys Calsitrans, Stomoxys Sitiens, Stomoxys Indica), Diptera: Muscidae.

Samples collected for PCR from recipient animals tested positive in 5 out of 6 cases, while the virus was isolated from 4 of 6 animals. The clinical signs exhibited by recipient animals were mostly moderate in nature with only one severe case. To our knowledge, this is the first time that transmission of LSDV by three Stomoxys species has been demonstrated, and their role as mechanical vectors of LSDV is indicated.

Development and evaluation of a novel real-time RT-PCR to detect foot-and-mouth disease viruses from the emerging A/ASIA/G-VII lineage.

A new lineage of foot-and-mouth disease virus (FMDV), called A/ASIA/G-VII, emerged from the Indian subcontinent in 2015 and continues to spread in Western Asia. Currently, the distribution of viruses belonging to this lineage is defined using sequencing approaches, but other cheaper and faster diagnostic methods are urgently needed. Thus, this study describes the development and validation of a novel A/ASIA/G-VII lineage-specific real-time RT-PCR (rRT-PCR). Diagnostic sensitivity and specificity were evaluated using representative field specimens and isolates from the A/ASIA/G-VII lineage, as well as samples comprising other FMDV lineages that co-circulate in Asia (n=54). This lineage-specific assay accurately detected all A/ASIA/G-VII samples tested (n=29), and no detection was observed for samples belonging to other FMDV lineages (n=25), namely A/ASIA/Sea-97, A/ASIA/Iran-05SIS-10, A/ASIA/Iran-05FAR-11, Asia1/ASIA/Sindh-08, O/CATHAY, O/ME-SA/PanAsia-2ANT-10, O/ME-SA/Ind-2001d, O/SEA/Mya-98. Additionally, the limit of detection was found to be at least equivalent to a pan-serotypic rRT-PCR assay. Therefore, these data indicate that this newly developed rRT-PCR assay can be applied to characterise field isolates in countries where the A/ASIA/G-VII lineage is endemic, as well as to monitor new incursions and outbreaks due to this lineage.