Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Sequential determination of viral load, humoral responses and phylogenetic analysis in fatal and non-fatal cases of Crimean-Congo hemorrhagic fever patients from Gujarat, India, 2019.

BACKGROUND: Thirty-four CCHF cases (17 fatal; 17 survived) were confirmed from Gujarat state, India during the year 2019. We aimed to find out the viral load, antibody kinetics, cytokine profile and phylogenetic analysis between fatal and non- fatal cases. METHODS: Thirty four cases were included in this study. Blood and urine samples were collected from all the cases on the day of admission to hospital. Non-fatal cases were followed weekly for understanding the profile of viral kinetics, anti-CCHFV IgM and IgG antibodies. We also quantified the cytokines in both fatal and non-fatal cases. For epidemiological correlation, livestock were screened for anti-CCHF IgG antibodies and the tick pool specimens were tested by real time RT-PCR. Virus isolation was attempted on tick pools and human specimens and phylogenetic analysis performed on human and ticks complete genome sequences. RESULTS: CCHF cases were detected throughout year in 2019 with the peak in August. Out of 34 cases, eight secondary CCHF cases were reported. Cases were predominantly detected in males and in 19-45 years age group (55.88%). The persistence of viremia was observed till 76th POD (post onset date) in one case whereas anti-CCHFV IgM and IgG was detected amongst these cases from the 2nd and 20th POD respectively. Positivity observed amongst livestock and tick pools were was 21.57% and 7.4% respectively. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. Fatal cases had high viral RNA copy numbers. Bleeding from one or two mucosal sites was significantly associated with fatality (OR-16.47;p-0.0034 at 95% CI). We could do CCHF virus isolation from two cases. Phylogenetic analysis revealed circulation of re-assortment of Asian-West African genotypes in humans and ticks. CONCLUSIONS: The persistence of CCHF viral RNA was detected till 76th POD in one of the survivors. The circulation of a re-assortment Asian-West African genotype in a CCHF case is also reported first time from India.

Foot and Mouth Disease (FMD) incidence in cattle and buffaloes and its associated farm-level economic costs in endemic India.

The study investigated the important epidemiological parameters and farm-level economic costs of FMD incidence in cattle and buffaloes during 2013-14 to 2015-16 in various states of India. Multistage random sampling procedure was adopted for the primary survey and data was collected through face-to-face personal interview from 18,609 cattle and buffalo rearing farm households from 123 districts across twelve states and one Union Territory. Besides epidemiological parameters, different farm-level direct and indirect loss associated with FMD was assessed at disaggregated level (states) by employing deterministic mathematical models. Highest number of affected villages and disease incidence was observed in non- FMD control programme (FMD-CP) implemented Madhya Pradesh and Assam states, respectively whereas negligible incidence was in FMD-CP implemented Punjab state. The disease incidence was high during 2013-14 and declined during 2014-15 and 2015-16, respectively implied severe incidence scenario (2013-14) succeeded by moderate (2014-15) and mild (2015-16) scenarios. The crossbred and high productive animals were severely affected than local breeds whereas on sexwise and agewise comparison revealed higher incidence in females and adult animals. During severe incidence scenario, milk loss/animal ranged from USD 6.87-47.44, 18.42-125.88, 16.33-91.43, and 27.17-123.62; mortality loss/animal ranged from USD 32.61-804.27, 30.76-577.7, 65.36-502.2, and 188.04-413.7; distress sale loss/animal ranged from USD 3.22-188.63, 64.34-519.3, 214.47-341.8, and 209.11-450.3; and opportunity cost of labour/animal from USD 5.49-54.29, 5.49-67.78; 7.95-31.37 and 9.83-72.38 in indigenous cattle, crossbred cattle, local and improved buffalo, respectively. The estimated draught power loss/animal varied from USD 39.46-142.94 with least being in Madhya Pradesh and highest in Assam states whereas the median treatment cost/animal was USD 9.18 and USD 27.07 in indigenous cattle and upgraded buffaloes, respectively. The total farm-level economic loss projected due to FMD in cattle and buffaloes in India was USD 3159 million (INR 221,110 million), USD 270 million (INR 18,910 million) and USD 152 million (INR 10,610 million), respectively during the severe, moderate and mild incidence scenarios at 2015-16 constant prices. The loss varied across the states, and in severe incidence scenario, the country might lose USD 3.2 billion/year and hence, the bi-annual vaccination schedule need to be strictly implemented in all the states. Besides timely vaccination coverage, managing unabated animal movement, educating and motivating the farmers to vaccinate their animals might reduce the incidence and consequential losses to various stakeholders in endemic states like India.

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018.

Equine glanders is an infectious and notifiable bacterial disease caused by Burkholderia mallei. The disease has been reported in South American, African and Asian countries including India. Here, we present the outcome of glanders serosurveillance carried out between January 2015 and December 2018 to know the status of equine glanders among different states in India. A total of 102,071 equid sera from 299 districts of twenty-one states and one union territory were tested for glanders. Samples were screened with Hcp1 indirect ELISA followed by confirmatory diagnosis by CFT. During this four-year surveillance, a total of 932 glanders-positive cases were detected from 120 districts of 12 states. The study also revealed increasing trend of glanders from 2016 onwards with maximum occurrence in northern India. Overall seroprevalence ranged between 0.62% (95% CI, 0.52-0.72) and 1.145% (95% CI, 1.03-1.25). Seasonal shifting from winter to summer (March to June) coincided with highest number glanders incidence with corresponding seroprevalences of 1.2% (95% CI, 1.09-1.30). The present surveillance unveils territorial ingression of glanders to six states like Jammu & Kashmir, Gujarat, Rajasthan, Madhya Pradesh, Delhi and Tamil Nadu. In addition, re-emerging cases have been reported in Maharashtra, Haryana and Punjab after a gap of 10 years. Lack of awareness, little veterinary care and unrestricted movement of equids across state borders might have led to the introduction and establishment of the infection to these states. We believe that information from this study will provide a baseline data on glanders for devising surveillance and control strategies in India. Being a zoonotic disease, the persistence of glanders poses a potential threat to occupationally exposed humans especially equine handlers and veterinarians. Therefore, targeted surveillance of human population from each glanders outbreak is also recommended.

Development of Multispecies Recombinant Nucleoprotein-Based Indirect ELISA for High-Throughput Screening of Crimean-Congo Hemorrhagic Fever Virus-Specific Antibodies.

Crimean-Congo hemorrhagic fever (CCHF) is a re-emerging zoonotic viral disease prevalent in many parts of Asia, Europe, and Africa. The causative agent, Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV), is transmitted through hard ticks. Tick vectors especially belonging to the Hyalomma species serve as the reservoir and amplifying host. The vertebrate animals including sheep, goat, and bovine act as a short-lasting bridge linking the virus and ticks. CCHFV causes fatal hemorrhagic fever in humans. Humans are usually infected with CCHFV either through the bite of infected ticks or by close contact with infected animals. Immunological assays, primarily enzyme-linked immunosorbent assay (ELISA) using whole viral antigen, are widely used for serosurveillance in animals. However, the whole virus antigen poses a high biohazard risk and can only be produced in biosafety level 4 laboratories. The present study focuses on the development and evaluation of safe, sensitive, and specific IgG indirect enzyme-linked immunosorbent assay (iELISA) using recombinant nucleoprotein (NP) of CCHF virus as an antigen. The codon-optimized NP gene sequence was synthesized, cloned, and expressed in pET28a+ vector. The recombinant NP was purified to homogeneity by affinity chromatography and characterized through Western blot and MALDI-TOF/MS analysis. The characterized protein was used to develop an indirect IgG microplate ELISA using a panel of animal sera. The in-house ELISA was comparatively evaluated vis-à-vis a commercially available ELISA kit (Vector-Best, Russia) with 76 suspected samples that revealed a concordance of 90% with a sensitivity and specificity of 79.4 and 100%, respectively. The precision analysis revealed that the assay is robust and reproducible in different sets of conditions. Further, the assay was used for serosurveillance in ruminants from different regions of India that revealed 18% seropositivity in ruminants, indicating continued circulation of virus in the region. The findings suggest that the developed IgG iELISA employing recombinant NP is a safe and valuable tool for scalable high-throughput screening of CCHFV-specific antibodies in multiple species.

Serosurvey of Crimean-Congo hemorrhagic fever virus in domestic animals, Gujarat, India, 2013.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral disease that causes a fatal hemorrhagic illness in humans. This disease is asymptomatic in animals. CCHF was first confirmed in a nosocomial outbreak in 2011 in Gujarat State. Another notifiable outbreak occurred in July, 2013, in Karyana Village, Amreli district, Gujarat State. Anti-CCHF virus (CCHFV) immunoglobulin G (IgG) antibodies were detected in domestic animals from the adjoining villages of the affected area, indicating a considerable amount of positivity against domestic animals. The present serosurvey was carried out to determine the prevalence of CCHFV among bovine, sheep, and goat populations from 15 districts of Gujarat State, India. A total of 1226 serum samples from domestic animals were screened for IgG antibodies using a CCHF animal IgG enzyme-linked immunosorbent assay (ELISA) kit from the Centers for Disease Control and Prevention. Antibodies were detected in all the 15 districts surveyed; with positivity of 12.09%, 41.21%, and 33.62% in bovine, sheep, and goat respectively. This necessitates the surveillance of CCHFV IgG antibodies in animals and hemorrhagic fever cases in human.

Descriptive epidemiology of equine influenza in India (2008-2009): temporal and spatial trends.

Equine influenza is a contagious viral disease that affects all members of the family Equidae, i.e., horses, donkeys and mules. The authors describe the pattern of equine influenza outbreaks in a number of states of India from July 2008 to June 2009. The disease was first reported in June 2008 in Katra (Jammu and Kashmir) and spread to ten other states within a year. All outbreaks of equine influenza in the various states were confirmed by laboratory investigations (virus isolation and/or serological confirmation based on haemagglutination inhibition [HI] assays of paired samples) before declaring them as equine influenza virus-affected state(s). The virus (H3N8) was reported from various locations in the country including Katra, Mysore (Karnataka), Ahmedabad (Gujarat), Gopeshwar and Uttarkashi (Uttarakhand) and was isolated in 9- to 11-day-old embryonated chicken eggs. The virus was confirmed as H3N8 by HI assays with standard serum and amplification of full-length haemagglutinin and neuraminidase genes by reverse transcriptase-polymerase chain reaction. Serum samples (n = 4 740) of equines from 13 states in India screened by HI revealed 1074 (22.65%) samples as being positive for antibodies to equine influenza virus (H3N8).