Understanding the tempo and mode of diversification and their consequence for tropical biodiversity remains a key challenge as different clades and regions exemplify distinct patterns and processes. We examined diversification rates and their drivers across 34 well-studied endemic lineages in peninsular India, one of the oldest regions of differentiation in the Oriental Realm, using birth-death models and their variations. We show that 18 lineages supported gradual species accumulation, suggesting that the historic stability of the landscape was an important driver. Additionally, paleotemperature, Miocene aridification, monsoon intensification and existing species diversity explained time-varying diversification patterns among the other 16 lineages. Net-diversification rates influenced differences in species diversity more than clade ages of peninsular Indian clades, where speciation rates mainly drove diversification, as extinction rates were low across taxa. Our results emphasise the importance of regional biogeographic, phylogenetic and geoclimatic history on the diversification dynamics among tropical landscapes.

In 2015, a novel highly divergent bovine calicivirus was detected in an Indian calf with enteritis. Phylogenetic analysis linked it to the Nebovirus with only 38.5% sequence identities, emphasizing the need for separate taxonomic classification. Furthermore, PCR screening detected these unique caliciviruses widely in India's northern states.

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Caliciviridae is a family of non-enveloped viruses distinguished by their single-stranded, positive-sense RNA genomes of 7.4–8.3 kb and characteristic "star-like" appearance under electron microscopy. Caliciviridae members cause a wide spectrum of illness in a broad range of animals and humans [1]. It currently encompasses eleven genera, seven of which (Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus, and Vesivirus) infect a wide variety of mammals, two (Bavovirus and Nacovirus) infect birds, and the remaining two (Minovirus and Salovirus) infect fish [1].

Enteritis in bovines has been linked to caliciviruses from two genera, Nebovirus and Norovirus [2-5]. However, Vesivirus infections have been found in bovines without causing enteritis [6]. We present the findings of a bovine enteric calicivirus, which is distantly related to neboviruses, in a calf with enteritis in India.

The Study

In the month of May of 2015, 14 cross-bred calves less than a year old reared in the temperate high-altitude Himalayan region (Mukteshwar, Uttarakhand, India), were presented with severe diarrhea, weight loss, and dehydration. To identify the causative pathogen, fecal samples (n = 14) were collected aseptically from the infected calves, all of which tested negative for genome of rotavirus group A (RVA) [7], Picobirnavirus (PBV) [8], Bovine Coronavirus (BCoV) [9], and Astrovirus (AstV) [10]. However, the RIDASCREEN Norovirus ELISA kit (R- Biopharm, Darmstadt, Germany) detected the possible presence of Norovirus in one of the samples, UK-B6. We archived this sample at -80 °C for future use. The next generation sequencing (NGS) was performed directly on the fecal sample at the ICMR-National Institute of

Virology, Pune, India and the complete genome sequence data retrieved was submitted to the National Centre for Biotechnology Information (NCBI) (GenBank accession no. MN241817).

The genome size of bovine calicivirus (UK-B6) retrieved from sample was 7,484 nt long, with a 91 nt long 5′ untranslated region (UTR), two open reading frames (ORF), ORF1 of 6,657 nt and ORF2 of 657 nt in length, and an 80 nt long 3′ UTR. ORF1 encoded a large polyprotein of 2,218 amino acids (aa) in length, which included non-structural proteins as well as a 542 aa long VP1 protein (capsid protein) at the 3′ end. ORF2 encoded a protein that was 218 aa long. The ORF1 polyprotein contained well-preserved amino acid motifs that are unique to caliciviruses (Appendix Table 1). Furthermore, the International Committee on Taxonomy of Viruses (ICTV) has established a criterion for demarcation of distinct genera within the Caliciviridae family, requiring that members in each genus should have more than 60% amino acid sequence divergence in their complete VP1 protein [1]. The VP1 protein of UK-B6 differs from all other caliciviruses by more than 60% in amino acid sequence including the prototypes of the Nebovirus (65.8% divergence with Newbury1_DQ013304 and 66.3% with Nebraska (NB)_AY082891), indicating that UK-B6 is a unique highly divergent calicivirus (Table 1). Furthermore, a search for VP1 amino acid similarity using Blastp algorithm (available at the NCBI) revealed that two caliciviruses, Kirklareli virus (YP_009272568) and BoNeV15/2021/CHN (WFD61513), had the highest amino acid identities with UK-B6, with 91.6% and 90.5%, respectively. These caliciviruses constitutes a new group (a possible new genus) differing from all other caliciviruses by >60% amino acid sequence differences in their complete VP1 protein.

Furthermore, the complete genome of Calcivirus from UK-B6 diverged significantly from existing member of Caliciviridae family’s genera. The whole genome sequence identities with the Nebovirus were the highest among all the Caliciviridae family genera, namely Newbury1 (38.5% nt, 36.9

A bovine population of approximately 302.79 million have a significant impact on the livelihoods of farmers in India. Nonetheless, various enteric pathogens pose a significant threat to these animals’ health, resulting in substantial economic losses. During the current study, we identified a novel bovine calicivirus in an Indian diarrheic calf that is distantly related to Nebovirus and meets the ICTV criteria for designation of a probable new genus. Although it has been demonstrated experimentally that Nebovirus prototypes such as Newbury-1 and Nebraska can cause enteritis, further research is required to determine the enteritis-causing potential of UK-B6 calicivirus detected in India. Moreover, a broader investigation into the prevalence of UK-B6 type viruses and 

their potential co-infections or associations with other pathogens responsible for enteritis is required.

Acknowledgments

The authors thank Dr. D.T. Maurya, Former Director of ICMR- NIV, Pune and Dr. R.K. Singh Former Director, ICAR-IVRI for the support.

Conflicts of Interest Statement

The authors have no conflicts of interest to declare.