First Report of Canine Bufavirus and Its Whole Genome Sequence in India

Canine bufavirus (CaBuV), a novel protoparvovirus of dogs was reported only in Italy and China, till date. It was detected from dogs with enteric and respiratory symptoms and is distantly related to the human bufavirus. To explore the enteric prevalence of CaBuV in India, 186 diarrheic faecal samples were collected in Telangana State between 2019 and 2020. Among the samples, 4.3% (8/186) were positive for CaBuV by PCR. Co-infection with canine parvovirus CPV-2 was seen in 75% (6/8) of CaBuV positive samples. The near complete genome (4292 bp) of CaBuV was amplified and reconstructed for one isolate 407/PVNRTVU/2020. Sequence alignment indicated 93.42–98.81% homology with the other available CaBuV sequences; 70.88-73.39%, and 54.4-54.8% identity with human bufavirus, and CPV-2, respectively. Phylogenetic analysis showed that CaBuV 407/PVNRTVU/2020 was most closely related to CaBuV Chinese strains, together separated as Asian lineage. All Bufaviruses clustered together in one clade; however, the bat and sea otter parvoviruses also showed close relation with Bufaviruses. This first report of prevalence of CaBuV in India provides a good reference, emphasizes the need for further epidemiological surveillance of CaBuV in India and its role in canine enteritis.

The Extrinsic Incubation Period of Zika Virus in Florida Mosquitoes Aedes aegypti and Ae. albopictus

The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread throughout the Americas. In July 2016, Florida experienced its first locally acquired ZIKV infection in the continental U.S. Concerns about health risks from ZIKV infection have increased the need to investigate the interactions between potential mosquito vectors and ZIKV. The time it takes for an arbovirus to propagate within a mosquito, and become transmissible, is the extrinsic incubation period (EIP). The EIP for potential mosquito vectors in Florida is unknown. To address this gap in the understanding of ZIKV epidemiology, Florida Aedes aegypti (L.) and Ae. albopictus (Skuse) were orally exposed to ZIKV infected blood meals and fully engorged mosquitoes were held at a constant temperature of 28 °C through the duration of the experiment. Saliva expectorates were collected from cohorts of mosquitoes and tested for the presence of ZIKV at three-day intervals over a period of 24 days to allow for an evaluation of the EIP of the emergent Asian lineage of ZIKV. High rates of infected bodies in Ae. albopictus (75–94%) and Ae. aegypti (68–86%) were observed throughout the incubation period, which did not differ by species. Higher rates of disseminated infection were observed later during the incubation period but did not differ between species. We calculated the 50% EIP to be shorter in Ae. albopictus than Ae. aegypti (16.2 and 18.2 days post infection, respectively). The competence for ZIKV observed in both species may contribute to high rates of ZIKV transmission in Florida populations.

Fossil wood of Syzygium from the Miocene of Guangxi, South China: the earliest fossil evidence of the genus in eastern Asia

A new species, Syzygium guipingensis sp. nov. (Myrtaceae), is described based on mummified fossil wood from the Miocene Erzitang Formation of Guiping Basin, Guangxi, South China. This species represents the most ancient reliable fossil record of the genus Syzygium in eastern Asia, showing the greatest similarity to the extant species S. buxifolium Hook. et Arnott. Its occurrence in the Miocene is consistent with the diversification age of the Asian lineage within Syzygium as estimated by molecular dating (11.4 Ma). The fossil record of Syzygium suggests that this genus migrated from Australia to eastern Asia in the Miocene, coincidently with the formation of island chains between these continents.

An epidemic Zika virus isolate suppresses antiviral immunity by disrupting antigen presentation pathways

Zika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.

African-lineage Zika virus replication dynamics and maternal-fetal interface infection in pregnant rhesus macaques

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in-vitro and in-vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titer and caused more severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here we infected four pregnant rhesus macaques with a low-passage strain of African-lineage ZIKV and compared its pathogenesis to a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. Viral replication kinetics were not significantly different between the two experimental groups and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1-1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV vRNA was found in maternal-fetal interface tissues in the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015-16. In its most recent update, the WHO stated that improved understanding of African-lineage pathogenesis during pregnancy must be a priority. Recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational non-human primate model. We show African-lineage isolates replicate with similar kinetics to Asian-lineage isolates and can infect the placenta. However, there was no evidence of more severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for future epidemiological and translational in-vivo studies with African-lineage ZIKV.

Reassessment of the Columnea latent viroid (CLVd) Taxonomic Classification

Columnea latent viroid (CLVd) is a member of the Pospiviroid family and its naked circular RNA genome typically forms native “rod-like” secondary structures. In this work, the CLVd taxonomy was reevaluated based on sequence similarity and phylogenetic analysis, as well as the evaluation of the symptom development and disease severity of four selected CLVd isolates in a range of host species. The phylogenetic analysis showed that all CLVd isolates were clustered into five distinct clades: (I) severe isolates originally found in tomato crops in Thailand, (II) ornamental isolates, (III) mild isolates originally found in tomato crops in Thailand, and two clades (IV and V) containing mild isolates originating mainly from tomato crops in European countries, with different virulence levels on several hosts. Our analysis demonstrated that some CLVd isolates have a sequence similarity of less than 90% within the species taxon, as well as distinct biological characteristics (symptom development and virulence), both of which are important ICTV criteria for viroid classification. For these reasons, we propose that CLVd should be re-classified into at least three main taxonomic lineages: a “CLVd-tomato Asian lineage” (I), a “CLVd-tomato European lineage” (IV) and a “CLVd-ornamental European lineage” (II), plus two minor lineages (III and V), fitting the ICTV criteria.

Nation-wide vector surveillance did not indicate transmission of the “American lineage pandemic ZIKA virus” in India

Background In wake of the global health emergency declared by the World Health Organization (WHO) during 2016, on the outbreak of ZIKA pandemic, Indian Council of Medical Research (ICMR) carried out countrywide vector surveillance for ZIKA and DENGUE viruses (ZIKV & DENV) in India, as a preparedness measure. Methods The study incorporated high-risk zones distributed to 49 Districts in 14 states/ Union Territories (UT) of India during 2016-2019. Seven ICMR Institutions undertook the study, following a uniform Standard Operating Protocol. Aedes specimens sampled on weekly intervals were processed by multiplex Reverse transcriptase PCR for ZIKV/DENV and Real time RT-PCR of ZIKV, among few samples distributed to all the Districts. Results Altogether, 79492 specimens of Aedes mosquitoes in 6492 pools were processed for both ZIKV and DENV infections. Among these, three and 63 pools respectively were found positive for ZIKV and DENV. ZIKV infections were recorded from Aedes aegypti sampled during 2018 sporadic ZIKA outbreak in Jaipur, Rajasthan, which belonged to the Asian lineage, already circulating in the Country. Both Ae. aegypti and Aedes albopictus were found infected with DENV and were distributed to ten states/ UTs. Both male and female specimens of Ae. albopictus recorded DENV infections indicating trans-ovarial transmission of DENV in the species. Conclusion This national vector surveillance study evinced no active transmission of the “American lineage - pandemic ZIKA virus” in India during 2016-2019, although Asian lineage of the virus already circulating in the Country was detected from Ae. aegypti from Jaipur, Rajasthan.

Kinetics of Asian and African Zika Virus Lineages over Single-cycle and Multi-cycle Growth in Culture: gene expression, cell killing, virus production, and mathematical modeling

Since 2014, an Asian lineage of Zika virus has caused outbreaks, and it has been associated with neurological disorders in adults and congenital defects in newborns. The resulting threat of the Zika virus to human health has prompted the development of new vaccines, which have yet to be approved for human use. Vaccines based on the attenuated or chemically inactivated virus will require large-scale production of the intact virus to meet potential global demands. Intact viruses are produced by infecting cultures of susceptible cells, a dynamic process that spans from hours to days and has yet to be optimized. Here, we infected Vero cells adhesively cultured in well-plates with two Zika virus strains: a recently isolated strain from the Asian lineage, and a cell-culture-adapted strain from the African lineage. At different time points post-infection, virus particles in the supernatant were quantified; further, microscopy images were used to quantify cell density and the proportion of cells expressing viral protein. These measurements were performed across multiple replicate samples of one-step infections every four hours over 60 hours and for multi-step infections every four to 24 hours over 144 hours, generating a rich dataset. For each set of data, mathematical models were developed to estimate parameters associated with cell infection and virus production. The African-lineage strain was found to produce a 14-fold higher yield than the Asian-lineage strain in one-step growth and a 7-fold higher titer in multi-step growth, suggesting a benefit of cell-culture adaptation for developing a vaccine strain. We found that image-based measurements were critical for discriminating among different models, and different parameters for the two strains could account for the experimentally observed differences. An exponential-distributed delay model performed best in accounting for multi-step infection of the Asian strain, and it highlighted the significant sensitivity of virus titer to the rate of viral degradation, with implications for optimization of vaccine production. More broadly, this work highlights how image-based measurements can contribute to discrimination of virus-culture models for the optimal production of inactivated and attenuated whole-virus vaccines.

Subgenomic flavivirus RNA (sfRNA) associated with Asian lineage Zika virus identified in three species of Ugandan bats (family Pteropodidae)

Serological cross-reactivity among flaviviruses makes determining the prior arbovirus exposure of animals challenging in areas where multiple flavivirus strains are circulating. We hypothesized that prior infection with ZIKV could be confirmed through the presence of subgenomic flavivirus RNA (sfRNA) of the 3′ untranslated region (UTR), which persists in tissues due to XRN-1 stalling during RNA decay. We amplified ZIKV sfRNA but not NS5 from three experimentally-infected Jamaican fruit bats, supporting the hypothesis of sfRNA tissue persistence. Applying this approach to 198 field samples from Uganda, we confirmed presence of ZIKV sfRNA, but not NS5, in four bats representing three species: Eidolon helvum (n = 2), Epomophorus labiatus (n = 1), and Rousettus aegyptiacus (n = 1). Amplified sequence was most closely related to Asian lineage ZIKV. Our results support the use of sfRNA as a means of identifying previous flavivirus infection and describe the first detection of ZIKV RNA in East African bats.