Molecular evolution and genotype shift of Porcine circoviruses type 2 in Vietnam.

Porcine Circovirus Type 2 (PCV2), a significant pathogen in the global swine industry, causes Porcine Circovirus Associated Diseases (PCVAD), contributing to substantial economic losses. This study investigates the genetic diversity and evolutionary dynamics of PCV2 in Vietnam from 2007 to 2023. We sequenced and analyzed 47 PCV2 genomes isolated from swine farms across Vietnam between 2022 and 2023, revealing predominant circulation of PCV2d (80.85%) followed by PCV2b (19.15%). Phylogenetic analysis identified PCV2 genotypes PCV2a, PCV2b, PCV2d, PCV2g, and PCV2h circulating in Vietnam, with PCV2d emerging as the most prevalent genotype. Comparison with historical data highlighted genotype shifts from PCV2b to PCV2d in 2014. Interestingly, PCV2h genotype was mainly observed between 2008 and 2012 but have not been detected since 2014. Regional analysis indicated varied PCV2 epidemiological patterns between northern and southern Vietnam. Amino acid substitutions within the capsid protein were identified, predominantly in antigenic regions critical for immune recognition. Positive selection analysis identified multiple sites under evolutionary pressure, indicating ongoing adaptation of Vietnamese PCV2 strains. These findings enhance understanding of PCV2 dynamics in Vietnam and underscore the importance of continuous surveillance and adaptive management strategies in controlling PCV2-associated diseases in swine populations.

Role of pre-existing immunity in driving the dengue virus serotype 2 genotype shift in the Philippines: A retrospective analysis of serological data.

OBJECTIVE: The invasion of dengue virus (DENV)-2 Cosmopolitan genotype into the Philippines, where the Asian II genotype previously circulated challenges the principle of dengue serotype-specific immunity. Assessment of antibodies in this population may provide a mechanistic basis for how new genotypes emerge in dengue-endemic areas. METHODS: We evaluated the neutralizing antibody (nAb) and antibody-dependent enhancement (ADE) responses against the two genotypes using archived serum samples collected from 333 patients with confirmed dengue in Metro Manila, Philippines, before, during, and after the introduction of the Cosmopolitan genotype. We quantified nAb titers in baby hamster kidney (BHK-21) cells with or without the Fcγ receptor IIA (FcγRIIA) to detect the capacity of virus-antibody complexes to neutralize or enhance DENV. RESULTS: The nAb potency of the archived serum samples against the two genotypes was greatly affected by the presence of FcγRIIA. We found significant differences in nAb titers between the two genotypes in BHK-21 cells with FcγRIIA (P <0.0001). The archived serum samples were incapable of fully neutralizing the Cosmopolitan genotype, but instead strongly promoted its ADE compared to the Asian II genotype (P <0.0001). CONCLUSION: These results reinforce the role of pre-existing immunity in driving genotype shifts. Our finding that specific genotypes exhibit differing susceptibilities to ADE by cross-reactive antibodies may have implications for dengue vaccine development.

Shift in dominant genotypes of Japanese encephalitis virus and its impact on current vaccination strategies.

Japanese encephalitis (JE) is a zoonotic ailment from the Japanese encephalitis virus (JEV). JEV belongs to the flavivirus genus and is categorized into a solitary serotype consisting of five genetically diverse genotypes (I, II, III, IV, and V). The JEV genotype III (GIII) was the prevailing strain responsible for multiple outbreaks in countries endemic to JEV until 1990. In recent years, significant improvements have occurred in the epidemiology of JE, encompassing the geographical expansion of the epidemic zone and the displacement of prevailing genotypes. The dominant genotype of the JEV has undergone a progressive shift from GIII to GI due to variations in its adaptability within avian populations. From 2021 to 2022, Australia encountered an epidemic of viral encephalitis resulting from infection with the GIV JEV pathogen. The current human viral encephalitis caused by GIV JEV is the initial outbreak since its initial discovery in Indonesia during the late 1970s. Furthermore, following a time frame of 50 years, the detection and isolation of GV JEV have been reported in Culex mosquitoes across China and South Korea. Evidence suggests that the prevalence of GIV and GV JEV epidemic regions may be on the rise, posing a significant threat to public safety and the sustainable growth of animal husbandry. The global approach to preventing and managing JE predominantly revolves around utilizing the GIII strain vaccine for vaccination purposes. Nevertheless, research has demonstrated that the antibodies generated by the GIII strain vaccine exhibit limited capacity to neutralize the GI and GV strains. Consequently, these antibodies cannot protect against JEV challenge caused by animal GI and GV strains. The limited cross-protective and neutralizing effects observed between various genotypes may be attributed to the low homology of the E protein with other genotypes. In addition, due to the GIV JEV outbreak in Australia, further experiments are needed to evaluate the protective efficiency of the current GIII based JE vaccine against GIV JEV. The alteration of the prevailing genotype of JEV and the subsequent enlargement of the geographical extent of the epidemic have presented novel obstacles in JE prevention and control. This paper examines the emerging features of the JE epidemic in recent years and the associated problems concerning prevention and control.

Detection and Complete Genome Analysis of Porcine Circovirus 2 (PCV2) and an Unclassified CRESS DNA Virus from Diarrheic Pigs in the Dominican Republic: First Evidence for Predominance of PCV2d from the Caribbean Region.

We report here high rates (47.5%, 48/101) of detection of porcine circovirus 2 (PCV2) in diarrheic pigs from three pig farms in the Dominican Republic. Seventeen of the PCV2 positive samples, representing the three pig farms, different age groups and sampling periods (2020-2021), were amplified for the complete PCV2 genome. Based on analysis of open reading frame 2 and complete genome sequences, the 17 PCV2 strains were assigned to the PCV2d genotype. Significant differences were observed in PCV2 detection rates between the vaccinated (20% (10/50)) and unvaccinated (62.5% (10/16) and 80% (28/35)) farms, corroborating previous observations that PCV2a-based vaccines confer protection against heterologous PCV2 genotypes. The present study is the first to report detection and molecular characterization of PCV2 from the Dominican Republic, warranting large-scale molecular epidemiological studies on PCV2 in pig farms and backyard systems across the country. For the first time, PCV2d was identified as the predominant PCV2 genotype in a study from the Caribbean region, suggesting that a genotype shift from PCV2b to PCV2d might be happening in the Caribbean region, which mirrored the current PCV2 genotype scenario in many other parts of the world. Besides PCV2, we also identified a pigeon circovirus-like virus, and a circular Replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus, which was characterized for the complete genome. The CRESS DNA virus shared a similar genomic organization and was related to unclassified CRESSV2 DNA viruses (belonging to the Order Cirlivirales) from porcine feces in Hungary, indicating that related unclassified CRESS DNA viruses are circulating among pigs in different geographical regions, warranting further studies on the epidemiology and biology of these novel viruses.

Genotype Shift of Malaysian Porcine Circovirus 2 (PCV2) from PCV2b to PCV2d within a Decade.

This paper aims to update the molecular status of porcine circovirus 2 (PCV2) in Malaysia. Firstly, the molecular detection rate of PCV2 in farm and sampled pig population were reported to be 83.78% (31/37 farms) and 83.54% (66/79 pigs) positive for PCV2, respectively. PCV2 was detected across all age groups, from fetuses, porkers to sows. Co-detection of PCV2 and PCV3 antigens was also reported at a rate of 28.77% (21/73). Secondly, PCV2 antigen was also detected in Malaysian abattoir lung samples: 18 out of 19 (94.74%) samples originating from clinically healthy finishers were tested positive. Further, this is the first study to confirm the circulation of PCV2 in the wild boar population roaming Peninsular Malaysia, where 28 out of 28 (100%) wild boar lung samples were found positive. One decade earlier, only genotype PCV2b was reported in Malaysia. This most recent update revealed that genotypes PCV2a, PCV2b and PCV2d were present, with PCV2d being the predominant circulating genotype. PCV2 cap gene nucleotide sequences in this study were found to be under negative selection pressure, with an estimated substitution rate of 1.102 × 10-3 substitutions/site/year (ssy).

Japanese encephalitis virus in India: An update on virus genotypes.

Japanese encephalitis (JE) is a leading cause of viral encephalitis in Southeast Asia. It is a serious public health issue in India, and cases have been emerging in newer areas of the country. Although vaccination efforts have already been initiated in the country since 2006 and later through the Universal Immunization Programme in 2011, still a significant reduction in the number of cases has to be achieved since an escalating trend of JE incidence has been reported in certain States such as Assam, Uttar Pradesh and West Bengal. Moreover, fresh cases of JE have been reported from certain pockets in Odisha as well. Despite the mass JE vaccination programme implemented in prioritized endemic zones in the country in 2011, a shift in the age group of JE virus (JEV) infection was noticed affecting the adult population in West Bengal. The recent detection of the circulation of genotype I (GI) in Gorakhpur, Uttar Pradesh and the co-circulation of GI and genotype III (GIII) in West Bengal are probably a warning signal for the public health personnel to strengthen the surveillance system in all endemic hotspots in the country. The abrupt emergence of JEV genotype V (GV) in China and Korea in 2009, after its first detection in Malaya in 1952, endemic countries have been cautioned to strengthen their surveillance, because GV has been suspected of getting dispersed efficiently in other parts of Asia. Moreover, the reduced protection efficiency of the JEV GIII-based vaccine against the JEV genotype V further warrants careful evaluation of the ongoing vaccination strategies in the endemic countries, anticipating the possible incursion of GV and its impact on future control strategies. In view of the above facts, the present communication reviews the current knowledge on the molecular epidemiology of JEV in India vis-a-vis the global scenario and discusses the future priorities in JEV research in India for effectively designing control strategies.

Potential Role of Birds in Japanese Encephalitis Virus Zoonotic Transmission and Genotype Shift.

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

Update of Genetic Diversity of Porcine Circovirus Type 2 in Chile Evidences the Emergence of PCV2d Genotype.

Porcine Circovirus 2 (PCV2) can cause multiple clinical conditions known as porcine circovirus-associated diseases (PCVAD). Before the wide availability of PCV2 vaccines, PCVAD resulted in significant losses to the global swine industry. PCV2's rapid evolutionary dynamics are comparable to single-stranded RNA viruses. Thus, shifts in the dominance and distribution of different genotypes may frequently occur, resulting in the emergence and spread of varying PCV2 genotypes and recombinant strains in swine. This study aims at identifying the PCV2 genotypes currently circulating in Chile. Seven hundred thirty-eight samples were obtained from 21 swine farms between 2020 and 2021. The samples were tested using PCR for species detection and genotyping. Sequencing and phylogenetic analyses were conducted in selected samples. PCV2 was detected in 26.9% of the PCR reactions and 67% of the sampled farms. The genotypes were determined in nine farms, PCV2a in one farm, PCV2b in four, and PCV2d in five, with PCV2b and PCV2d co-circulating in one farm. The phylogenetic analysis of twelve ORF2 sequences obtained (PCV2a = 5; PCV2b = 4; PCV2d = 3) showed a PCV2a Chilean strains monophyletic cluster; closely related to Chilean viruses collected in 2012 and 2013. Of the three different PCV2b sequenced viruses, two viruses were close to the root of the PCV2b group, whereas the remaining one grouped with a South Korean virus. PCV2d sequences were closely related to Asian viruses. A previously reported PCV2a/PCV2d recombinant strain was not detected in this study. Our results suggest the emergence and potential shift to PCV2d genotype in Chilean farms.

Genetic Diversity of Porcine Circovirus Isolated from Korean Wild Boars.

In Korea, three genotypes of porcine circovirus type 2 (PCV2a, PCV2b, and PCV2d) have been identified on domestic pig farms, while two genotypes (PCV2a and PCV2b) have been identified in wild boar populations. Here, we investigated genotype diversity and genotypic shift in 91 PCV2 isolates from 1340 wild boars captured in South Korea between 2013 and 2017. Phylogenetic analyses based on the complete ORF2 showed that the 91 PCV2 strains were detected as four genotypes by qPCR screening assay: PCV2a (2.2%, 2/91), PCV2b (16.5%, 15/91), PCV2d (80.2%, 73/91), and PCV2h (1.1%, 1/91). Only one intergenotype recombinant event was detected between PCV2 ORF2 in wild boars (PCV2b) and domestic pigs (PCV2a). Amino acid positions 86-89 within ORF2, which distinguishes the different genotypes, were conserved in all PCV2 genotypes isolated from South Korean wild boars, including TNKI in PCV2a/PCV2h, SNPR in PCV2b, and SNPL in PCV2d. The estimated nucleotide substitution rates in the ORF2 region of viruses from South Korean wild boars and domestic pigs were 5.8145 × 10-4 and 4.5838 × 10-4 substitutions per site per year (s/s/y), respectively. The times to the most recent common ancestor (tMRCA) for South Korean domestic pig PCV2 were 1937 (PCV2a), 1972 (PCV2b), 1999 (PCV2d-1), and 2000 (PCV2d-2). By contrast, the tMRCA for South Korean wild boar PCV2b and PCV2d were 1989 and 2001, respectively. Thus, the PCV2d genotype is prevalent among South Korean wild boars and domestic pigs.

Porcine circovirus type 2 (PCV2) genotyping in Austrian pigs in the years 2002 to 2017.

BACKGROUND: Eight different PCV2 genotypes with varying prevalence and clinical impact have been described so far. PCV2 infection is still widespread among the vaccinated population and several experimental studies have clearly demonstrated that there is no induction of a 100% cross-protective immunity between the PCV2 genotypes. Hence, PCV2a-based vaccines may be ineffective. In this longitudinal study, the PCV2 genotype and haplotype evolution in Austria in the years 2002 to 2017 was investigated by phylogenetic analysis of 462 bp-long sequences of the capsid protein gene (ORF2). The obtained findings may be of practical relevance for the future development of vaccination strategies. RESULTS: One hundred thirty four of a total of 161 formalin-fixed and paraffin wax-embedded samples could be sequenced successfully. There was no significant influence of storage time on sequencing success or quality. PCV2a (8.2%), PCV2b (77.6%), PCV2d (13.4%), and PCV2g (0.8%) were found. PCV2d was first detected as early as in 2004. PCV2g was described once in 2009. Both global PCV2 genotype shifts were observed. PCV2a occurred with a low prevalence during the first study years only in samples from non-vaccinated swine herds and was gradually replaced by PCV2b until 2011. PCV2b was the most prevalent genotype over the whole study period and was detected in samples from vaccinated and non-vaccinated herds. During the last two study years, the prevalence of PCV2d increased, although at this point almost all herds were vaccinated. The haplotype diversity was high, but the nucleotide diversity was low. Especially for genotype PCV2b, an increase in haplotype diversity could be described during the first study years. CONCLUSION: Extensive PCV2a-derived vaccination resulted in a reduction of prevalence and in a stabilization of genotype PCV2a, whereas genotypes PCV2b and PCV2d evolved as a consequence of natural and vaccination-induced selection. An ongoing virus circulation may be the result of reduced vaccine-induced protection.

Genotypic diversity of porcine circovirus type 2 (PCV2) and genotype shift to PCV2d in Korean pig population.

Porcine circovirus type 2 (PCV2) is a causative agent of PCV2-associated disease (PCVAD), which leads to enormous economic losses in the swine industry worldwide. A high nucleotide substitution rate allows for the continuous evolution of PCV2 and the emergence of novel PCV2 strains. However, the distribution of emerging PCV2 genotypes and the co-existence of multiple genotypes in Korea have not been elucidated. The objective of this study was to determine the genetic diversity of PCV2 in Korean pig herds between 2009 and 2016. The overall prevalence of PCV2, from various samples originating from commercial pigs, was 53.8% (325/604). Two cases of a genotype shift to PCV2d at the farm level showed that the genotype shift started before 2012. In addition, genotype-specific PCR, on pen-based oral fluid samples for nationwide PCV2 surveillance in 2016, indicated that the infection pattern of PCV2 genotypes at the farm level was as follows; none (6/69), PCV2a (6/69), PCV2b (2/69), PCV2d (33/69), PCV2a/b (2/69), PCV2a/d (4/69), PCV2b/d (11/69), and PCV2a/b/d (2/69), respectively. This suggests that the genotype shift to PCV2d occurred on a nationwide scale and that the co-existence of different genotypes is common in Korean pig herds. In addition, seven sites on the capsid protein of Korean PCV2 were identified as being under positive selection pressure, all of which are related to the epitope region and neutralization activity. These data provide evidence of increased genetic diversity and shifts among Korean PCV2 isolates.

Continuous dengue type 1 virus genotype shifts followed by co-circulation, clade shifts and subsequent disappearance in Surabaya, Indonesia, 2008-2013.

Four serotypes of dengue virus (DENV-1 to DENV-4) and their genotypes are distributed in tropical and subtropical regions. Indonesia has been recently suggested as the origin of some dengue virus genotypes. In Surabaya, the second biggest city of Indonesia, we previously reported a shift of the predominantly circulating serotype from DENV-2 to DENV-1 in November 2008, followed by a genotype shift of DENV-1 from genotype IV (GIV) to genotype I (GI) in September 2009, based on nucleotide sequences in the envelope protein coding region. Since then, GI strains had predominantly circulated until December 2010. In this report, we investigated further DENV-1 transitions in Surabaya during 2011-2013 in order to comprehend dengue dynamics during 2008-2013 in more detail. From January 2011 through December 2011, only GIV strains were isolated, indicating that a genotype shift again took place from GI to GIV. In January 2012, GI and GIV strains started co-circulating, which continued until June 2013. To further investigate this phenomenon, analysis was performed at a clade level. GI and GIV strains isolated in Surabaya formed four and three distinct clades, respectively. Concomitant with co-circulation, new clade strains appeared in both genotypes. In contrast, some previously circulating clades were not isolated during co-circulation, indicating clade shifts. Among our Surabaya isolates, nucleotide and amino acid differences in the E region were, respectively, 1.0-2.3% and 0.2-1.0% for GI isolates and 2.0-6.3% and 0.0-1.8% for GIV isolates. Several characteristic amino acid substitutions in the envelope ectodomain were observed in some clades. After July 2013, DENV-1 strains were not isolated and were replaced with DENV-2. This study showed that continuous shifts of more than one genotype resulted in their co-circulation and subsequent disappearance and suggested the relevance of clade replacement to genotype co-circulation and disappearance in Surabaya.